U.S. patent application number 13/266794 was filed with the patent office on 2012-07-19 for treatment of diseases with altered smooth muscle contractility.
Invention is credited to Jeanine D'Armiento, Andrew Marks, Steven Marx.
Application Number | 20120184517 13/266794 |
Document ID | / |
Family ID | 43032732 |
Filed Date | 2012-07-19 |
United States Patent
Application |
20120184517 |
Kind Code |
A1 |
Marx; Steven ; et
al. |
July 19, 2012 |
TREATMENT OF DISEASES WITH ALTERED SMOOTH MUSCLE CONTRACTILITY
Abstract
The present invention provides, inter alia, methods and
compositions for treating or ameliorating the effects of a disease
characterized by altered smooth muscle contractility, such as e.g.,
asthma.
Inventors: |
Marx; Steven; (Scarsdale,
NY) ; D'Armiento; Jeanine; (New York, NY) ;
Marks; Andrew; (Larchmont, NY) |
Family ID: |
43032732 |
Appl. No.: |
13/266794 |
Filed: |
April 30, 2010 |
PCT Filed: |
April 30, 2010 |
PCT NO: |
PCT/US10/01288 |
371 Date: |
March 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61214948 |
Apr 30, 2009 |
|
|
|
Current U.S.
Class: |
514/171 ;
514/456 |
Current CPC
Class: |
A61P 11/06 20180101;
A61P 21/00 20180101; A61K 31/352 20130101; A61P 29/00 20180101;
A61K 36/47 20130101; A61P 11/00 20180101; A61P 9/12 20180101; A61K
31/00 20130101; A61P 13/10 20180101; A61K 9/0078 20130101; A61K
45/06 20130101; A61K 31/352 20130101; A61K 2300/00 20130101; A61K
36/47 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/171 ;
514/456 |
International
Class: |
A61K 31/56 20060101
A61K031/56; A61P 11/06 20060101 A61P011/06; A61P 21/00 20060101
A61P021/00; A61P 9/12 20060101 A61P009/12; A61P 13/10 20060101
A61P013/10; A61P 29/00 20060101 A61P029/00; A61K 31/352 20060101
A61K031/352; A61P 11/00 20060101 A61P011/00 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under P01
HL081172 awarded by the National Heart, Lung and Blood Institute of
the National Institutes of Health. The government has certain
rights in the invention.
Claims
1. A method of treating or ameliorating the effects of a disease
characterized by altered smooth muscle contractility comprising
administering to a patient suffering from such a disease an
effective amount of a large-conductance Ca.sup.2+-activated K.sup.+
(BK) channel modulator.
2. The method according to claim 1, wherein the BK channel
modulator is a BK channel activator.
3. The method according to claim 2, wherein the BK channel
activator is selected from the group consisting of rottlerin,
flindokalner (Bristol-Myers Squibb), BMS-554216 (Bristol-Myers
Squibb), Pharmaprojects No. 4420 (Merck & Co), Pharmaprojects
No. 4494 (Merck & Co), NS-1619 (NeuroSearch), NSD-551
(NeuroSearch), NS-8 (Nippon Shinyaku), a pharmaceutically
acceptable salt thereof, and combinations thereof.
4. The method according to claim 3, wherein the BK channel
activator is rottlerin.
5. The method according to claim 4, wherein the rottlerin is in the
form of an extract from Mallotus phillippinensis.
6. The method according to claim 1, wherein the disease is selected
from the group consisting of asthma, chronic obstructive pulmonary
disease, urinary incontinence, and hypertension.
7. The method according to claim 1, wherein the disease is
asthma.
8. The method according to claim 1, wherein the disease is
chronic.
9. The method according to claim 1, wherein the disease is
acute.
10. The method according to claim 1, wherein the BK channel
modulator is administered as part of a pharmaceutical
composition.
11. The method according to claim 10, wherein the pharmaceutical
composition is in a unit dosage form.
12. The method according to claim 11, wherein the unit dosage form
is inhaled.
13. The method according to claim 10, wherein the pharmaceutical
composition is co-administered with a composition selected from the
group consisting of corticosteroids, anti-cholinergics,
anti-leukotrienes, .beta.-agonists, and phosphodiesterase
inhibitors.
14. The method according to claim 13, wherein the pharmaceutical
composition is co-administered with a .beta.-agonist.
15. A method of treating or ameliorating the effects of asthma
comprising administering to a patient suffering from asthma an
effective amount of a BK channel modulator.
16. The method according to claim 15, wherein the BK channel
modulator is administered as part of a pharmaceutical
composition.
17. The method according to claim 16, wherein the pharmaceutical
composition is in a unit dosage form.
18. The method according to claim 17, wherein the unit dosage form
is inhaled.
19. The method according to claim 16, wherein the pharmaceutical
composition is co-administered with a composition selected from the
group consisting of corticosteroids, anti-cholinergics,
anti-leukotrienes, .beta.-agonists, and phosphodiesterase
inhibitors.
20. The method according to claim 19, wherein the pharmaceutical
composition is co-administered with a .beta.-agonist.
21. A method for decreasing airway constriction and/or airway
resistance in a patient without increasing the heart rate of the
patient comprising administering to the patient an effective of
amount of a BK channel modulator or a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a BK channel
modulator.
22. The method according to claim 21, wherein the pharmaceutical
composition is in a unit dosage form.
23. The method according to claim 22, wherein the unit dosage form
is inhaled.
24. The method according to claim 21, wherein the pharmaceutical
composition is co-administered with a composition selected from the
group consisting of corticosteroids, anti-cholinergics,
anti-leukotrienes, .beta.-agonists, and phosphodiesterase
inhibitors.
25. The method according to claim 24, wherein the pharmaceutical
composition is co-administered with a .beta.-agonist.
26. A method for modulating inflammation in a lung of a patient,
the method comprising administering to a patient an effective of
amount of a BK channel modulator or a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a BK channel
modulator, which amount is sufficient to modulate the
inflammation.
27. The method according to claim 26, wherein the modulation is a
decrease in inflammation.
28. The method according to claim 26, wherein the pharmaceutical
composition is in a unit dosage form.
29. The method according to claim 28, wherein the unit dosage form
is inhaled.
30. The method according to claim 26, wherein the pharmaceutical
composition is co-administered with a composition selected from the
group consisting of corticosteroids, anti-cholinergics,
anti-leukotrienes, .beta.-agonists, and phosphodiesterase
inhibitors.
31. The method according to claim 30, wherein the pharmaceutical
composition is co-administered with a .beta.-agonist.
32. A pharmaceutical composition for treating or ameliorating the
effects of a disease characterized by altered smooth muscle
contractility, the composition comprising a pharmaceutically
acceptable carrier and a BK channel modulator.
33. The pharmaceutical composition according to claim 32, wherein
the disease is selected from the group consisting of asthma,
chronic obstructive pulmonary disease, urinary incontinence, and
hypertension.
34. The pharmaceutical composition according to claim 32, wherein
the disease is asthma.
35. The pharmaceutical composition according to claim 32, which is
in a unit dosage form.
36. The pharmaceutical composition according to claim 35, wherein
the unit dosage form is inhaled.
37. The pharmaceutical composition according to claim 32, which is
co-administered with a composition selected from the group
consisting of corticosteroids, anti-cholinergics,
anti-leukotrienes, .beta.-agonists, and phosphodiesterase
inhibitors.
38. The method according to claim 37, wherein the pharmaceutical
composition is co-administered with a .beta.-agonist.
39. A pharmaceutical composition for treating or ameliorating the
effects of asthma, the composition comprising a pharmaceutically
acceptable carrier and a BK channel modulator.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit to U.S. Provisional Patent
Application Ser. No. 61/214,948, filed Apr. 30, 2009, the entire
content of which is hereby incorporated by reference as if recited
in full herein.
FIELD OF THE INVENTION
[0003] The present invention relates, inter alia, to pharmaceutical
compositions and methods to treat or ameliorate the effects of
diseases characterized by altered smooth muscle contractility, such
as e.g., asthma.
BACKGROUND OF THE INVENTION
[0004] Asthma-associated airway hyperresponsiveness (AHR) is
primarily mediated by excessive airway smooth muscle (ASM) cell
contraction, yet the mechanisms responsible for this behavior are
not clearly elucidated. Although asthma involves inflammation, ASM
cell hypertrophy and hyperplasia, the primary event leading to AHR
is the stimulation of ASM cell contraction. Despite current therapy
(anti-cholinergics, anti-histamines, anti-leukotrienes,
.beta.-agonists and phosphodiesterase inhibitors), many asthmatic
patients suffer from airway hyperreactivity. In addition,
side-effects from these drugs can also limit their efficacy. Thus,
novel approaches to treat asthma may have a profound impact on
improving the morbidity of this disease. Regulating the growth and
contractility of ASM represents an important target for the
treatment of asthma.
[0005] The elevation of intracellular calcium, [Ca.sup.2+].sub.i,
which may occur in asthma (Black et al., Intrinsic asthma: is it
intrinsic to the smooth muscle? Clin Exp Allergy (2009); Kellner et
al., Mechanisms altering airway smooth muscle cell Ca+ homeostasis
in two asthma models. Respiration 76:205-215. (2008)), plays a
critical role in airway smooth muscle (ASM) contractility and may
also affect cell proliferation. The increase in Ca.sup.2+ can be
achieved in two ways: (a) release of Ca.sup.2+ from the internal
stores of the SR and/or (b) Ca.sup.2+ influx from the extracellular
space via plasma membrane ion channels. Contraction of smooth
muscle is triggered by phosphorylation of myosin, catalyzed by
Ca.sup.2+/calmodulin-dependent myosin light chain kinase (MLCK),
which is activated by Ca.sup.2+. In airway and vascular SMCs
agonists initiate, but cannot maintain, contraction in
Ca.sup.2+-free conditions, which indicates that internal stores
require refilling by Ca.sup.2+ influx. The Ca.sup.2+ influx may be
mediated by voltage-dependent and voltage-independent mechanisms.
The contractility of smooth muscle is regulated by a feed-back
mechanism whereby the localized, transient increase in cytoplasmic
Ca.sup.2+ concentration due to activation of sarcoplasmic reticular
(SR) ryanodine receptors (RyR) activates plasma membrane BK
channels (large conductance voltage- and Ca.sup.2+-activated
K.sup.+ channels). The activation of BK channels causes transient
membrane hyperpolarization, inhibition of Ca.sup.2+ influx through
voltage-dependent Ca.sup.2+ channels, reduced intracellular
Ca.sup.2+ concentration ([Ca.sup.2+].sub.i and a subsequent
decrease in smooth muscle tension.
[0006] Furthermore, the high incidence of stroke and hypertension
in the United States remains a leading indication for visits to
physicians, the use of prescription drugs and morbidity/mortality.
It is estimated that more than 50 million Americans (approximately
one third of the adult population) currently suffer from
hypertension. Two-thirds of the population over age 70 suffers from
hypertension. Chronic blood pressure elevation leads to end-organ
damage, including eye, cardiac, and central nervous system damage.
Thus, greater understanding of the molecular mechanisms leading to
the regulation of membrane excitability may have important
implications in improving therapeutic modalities.
[0007] The large-conductance Ca.sup.2+-activated K.sup.+
(BK.sub.Ca) channel complex plays a critical role in regulating
contractile tone in smooth muscle and the vasculature (Brenner, et
al., Vasoregulation by the .beta.1 subunit of the calcium-activated
potassium channel. Nature, 2000. 407(6806):870-6; Brayden, et al.,
Regulation of arterial tone by activation of calcium-dependent
potassium channels. Science, 1992. 256(5056):532-5). Furthermore,
neuronal BK.sub.Ca channel function is not well-studied, yet it
remains clear that the channel has significant effects on
neurotransmitter release and neuronal discharges (Robitaille, et
al., Functional colocalization of calcium and calcium-gated
potassium channels in control of transmitter release. Neuron, 1993.
II(4):645-55). Thus, the BK.sub.Ca channel represents an important
integrator of signal transduction pathways, potently mediating
cellular excitability in a diverse group of cell types. Recent
studies have suggested that the channel may have a role in innate
immunity in neutrophils (Ahluwalia, et al., The large-conductance
Ca.sup.2+-activated K.sup.+ channel is essential for innate
immunity. Nature, 2004. 427(6977):853-8), recognition as a
heme-binding protein (Tang, et al., Haem can bind to and inhibit
mammalian calcium-dependent Slo1 BK channels. Nature, 2003.
425(6957):531-5), behavior responses to ethanol (Davies, et al., A
central role of the BK potassium channel in behavioral responses to
ethanol in C. elegans. Cell, 2003. 115(6):655-66) and function as a
protective mechanism against ischemically-driven cell death in
cardiac myocytes (Xu, et al., Cytoprotective role of
Ca.sup.2+-activated K.sup.+ channels in the cardiac inner
mitochondrial membrane. Science, 2002. 298(5595):1029-33). These
novel functions of the BK.sub.Ca channel remain to be further
validated and explored (Mocaydlowski, E. G., BK Channel News: Full
Coverage on the Calcium Bowl. J. Gen. Physiol, 2004. 123(5):471-3).
Utilizing molecular biologic and electrophysiologic approaches, the
present inventors are seeking to elucidate the mechanism(s) through
which the BK.sub.Ca channel is allosterically regulated.
[0008] By unraveling the complex mechanism(s) mediating
phosphorylation-dependent and .beta.1 subunit regulation of the
channel, the present inventors seek to identify specific regions
that are responsible for activation and inhibition of channel
function. Novel approaches for the treatment of disorders ranging
from neurologic dysfunction (seizures, memory) to vascular
complications related to diabetes or hypertension will follow from
elucidation of the basic mechanism(s) mediating BK.sub.Ca channel
activity.
Regulation of Blood Pressure and Vascular Smooth Muscle Cell (VSMC)
Contractility by Ca.sub.v1.2, Ryanodine Receptor (RyR) and
BK.sub.Ca Channels
[0009] Arterial blood pressure is determined by several factors,
including vascular tone, which represents the contractile activity
of smooth muscle within the walls of resistance vessels. The
contractile state of smooth muscle is organized through the
interplay of vasoconstrictor and vasodilatory neurohormones and by
blood pressure itself (the Bayliss effect; constriction of the
vessel after an increase in transmural pressure) (Bayliss, W. M.,
On the local reactions of the arterial wall to changes of internal
pressure. J. Physiol., 1902. 28:220-23; Nelson, M. T., Bayliss,
myogenic tone and volume-regulated chloride channels in arterial
smooth muscle. J. Physiol., 1998. 507 (Pt 3):629; Nelson, et al.,
Noradrenaline contracts arteries by activating voltage-dependent
calcium channels. Nature, 1988. 336(6197):382-5). The
autoregulatory Bayliss effect is based upon graded membrane
depolarization in response to pressure, which activates voltage
dependent Ca.sup.2+ channels, causing vasoconstriction. Vascular
smooth muscle contraction is triggered by Ca.sup.2+/calmodulin
dependent phosphorylation of the regulatory myosin light chain.
Increased intracellular Ca.sup.2+ is mediated by Ca.sup.2+ influx
through Ca.sub.v1.2 and Ca.sup.2+ release from intracellular
stores, mainly through the Inositol 1,4,5-Triphosphate Receptor
(IP3R) (Davis, et al., Signaling mechanisms underlying the vascular
myogenic response. Physiol. Rev., 1999. 79(2):387-423). Ca.sub.v1.2
was recently reported to play a critical role in regulating smooth
muscle contraction/blood pressure regulation, as an inducible
smooth muscle specific Ca.sub.v1.2 knockout demonstrated abnormal
autoregulation and maintenance of vascular tone in response to
depolarization and pressure (Moosmang, et al., Dominant role of
smooth muscle L-type calcium channel Ca.sub.v1.2 for blood pressure
regulation. Embo. J., 2003. 22(22):6027-34). In vascular smooth
muscle, the dynamic range of intracellular calcium concentrations,
[Ca.sup.2+].sub.i, is narrow, ranging from .about.100 nM when the
artery is maximally dilated to 350 nM when arteries are maximally
constricted (Knot, et al., Regulation of arterial diameter and wall
[Ca.sup.2+] in cerebral arteries of rat by membrane potential and
intravascular pressure. J. Physiol, 1998. 508:199-209).
[0010] Spontaneous transient outward currents (STOC) were first
described in smooth muscle by Bolton and coworkers (Benham, et al.,
Spontaneous transient outward currents in single visceral and
vascular smooth muscle cells of the rabbit. J Physiol, 1986.
381:385-406; Bolton, et al., Spontaneous transient outward currents
in smooth muscle cells. Cell Calcium, 1996. 20(2):141-52) and have
been shown in a diverse group of vascular and non-vascular smooth
muscle (Hisada, et al., Properties of membrane currents in isolated
smooth muscle cells from guinea-pig trachea. Pflugers Arch., 1990.
416(1-2):151-61; Ohya, et al., Cellular calcium regulates outward
currents in rabbit intestinal smooth muscle cell. Am. J. Physiol,
1987. 252(4 Pt I):C401-10; Saunders, et al., Spontaneous transient
outward currents and Ca.sup.++-activated K.sup.+ channels in swine
tracheal smooth muscle cells. J. Pharmacol. Exp. Ther., 1991.
257(3): 1114-20; Nelson, et al., Relaxation of arterial smooth
muscle by calcium sparks. Science, 1995. 270(5236):633-637; Nelson,
et al., Physiological roles and properties of potassium channels in
arterial smooth muscle. Am. J. Physiol, 1995. 268 (Cell Physiol.
37):C799-C822; Hume, et al., Macroscopic K.sup.+ currents in single
smooth muscle cells of the rabbit portal vein. J. Physiol, 1989.
413:49-73; Jaggar, et al., Ca.sup.2+ channels, ryanodine receptors
and Ca.sup.2+-activated K.sup.+ channels: a functional unit for
regulating arterial tone. Acta Physiol. Scand., 1998.
164(4):577-87; Porter, et al., Frequency modulation of Ca.sup.2+
sparks is involved in regulation of arterial diameter by cyclic
nucleotides. Am. J. Physiol, 1998. 274(5 Pt I):C1346-55). Each
transient outward current represents the activation of 10-100
BK.sub.Ca channels (Porter, et al., Frequency modulation of
Ca.sup.2+ sparks is involved in regulation of arterial diameter by
cyclic nucleotides. Am. J. Physiol, 1998. 274(5 Pt I):C1346-55).
Nelson and colleagues obtained the first evidence of Ca.sup.2+
sparks in smooth muscle (Nelson, et al., Relaxation of arterial
smooth muscle by calcium sparks. Science, 1995. 270(5236):633-637)
and similar findings have been shown in numerous smooth muscle
cells derived from arteries, portal vein, urinary bladder,
gastrointestinal tract, airway and gallbladder (Mironneau, et al.,
Ca.sup.2+ sparks and Ca.sup.2+ waves activate different
Ca.sup.2+-dependent ion channels in single myocytes from rat portal
vein. Cell Calcium, 1996. 20(2): 153-60; Gordienko, et al.,
Crosstalk between ryanodine receptors and IP3Rs as a factor shaping
spontaneous Ca.sup.2+-release events in rabbit portal vein
myocytes. J. Physiol., 2002. 542(Pt 3):743-62; Herrera, et al.,
Voltage dependence of the coupling of Ca.sup.2+ sparks to BK.sub.Ca
channels in urinary bladder smooth muscle. Am. J. Physiol. Cell
Physiol, 2001. 280(3):C481-90; Ji, et al., Stretch-induced calcium
release in smooth muscle. J. Gen. Physiol, 2002. 119(6):533-44; Ji,
et al., RYR2 proteins contribute to the formation of Ca.sup.2+
sparks in smooth muscle. J. Gen. Physiol, 2004. 123(4):377-86;
Gordienko, et al., Variability in spontaneous subcellular calcium
release in guinea-pig ileum smooth muscle cells. J. Physiol, 1998.
507 (Pt 3):707-20; Kirber, et al., Relationship of Ca.sup.2+ sparks
to STOCs studied with 2D and 3D imaging in feline oesophageal
smooth muscle cells. J. Physiol, 2001. 531(Pt 2):315-27). Ca.sup.2+
sparks are transient local increases in intracellular Ca.sup.2+
that occur through the coordinated opening of a group of RyR
located on the SR (Nelson, et al., Relaxation of arterial smooth
muscle by calcium sparks. Science, 1995. 270(5236):633-637). In
cerebral artery myocytes, Ca.sup.2+ sparks lead to activation of
the BK.sub.Ca channel, thus providing an important feedback role in
the regulation of pressure-induced constriction (Nelson, et al.,
Relaxation of arterial smooth muscle by calcium sparks. Science,
1995. 270(5236):633-637). Vasodilators may act, in part, through
increasing the frequency of Ca.sup.2+ sparks.
[0011] All three RyR isoforms have been reported in smooth muscle
(Marks, et al., Molecular cloning and characterization of the
ryanodine receptor/junctional channel complex cDNA from skeletal
muscle sarcoplasmic reticulum. Proc. Natl Acad. Sci., 1989.
86:8683-8687; Hakamata, et al., Primary Structure and distribution
of a novel ryanodine receptor/calcium release channel from rabbit
brain. FEBS, 1992. 312:229-235; Ledbetter, et al., Tissue
distribution of ryanodine receptor isoforms and alleles determined
by reverse transcription polymerase chain reaction. Journal of
Biological Chemistry, 1994. 269(50):31544-51) although the relative
proportion of each isoform varies between tissues (Xu, et al.,
Evidence for a Ca.sup.2+-gated ryanodine-sensitive Ca.sup.2+
release channel in visceral smooth muscle. Proc. Natl. Acad. Sci.
USA, 1994. 91(8):3294-8). The physiologic role of each of the
isoforms of the RyR is lacking. The respective roles of RyR2 and
RyR1 in smooth muscle have been incompletely elucidated (Takeshima,
et al., Excitation-contraction uncoupling and muscular degeneration
in mice lacking functional skeletal muscle ryanodine-receptor gene.
Nature, 1994. 369(6481):556-9; Takeshima, et al., Ca.sup.2+-induced
Ca.sup.2+ release in myocytes from dyspedic mice lacking the type-1
ryanodine receptor. Embo. J., 1995. 14(13):2999-3006), in part,
because RyR2 null mice are lethal (Takeshima, et al., Embryonic
lethality and abnormal cardiac myocytes in mice lacking ryanodine
receptor type 2. Embo. J., 1998. 17(12):3309-16). In rat portal
vein myocytes, antisense oligonucleotides targeting each of the RyR
isoforms demonstrated that both RyR1 and RyR2 are required for
myocytes to respond to membrane depolarization with Ca.sup.2+
sparks and global increase in intracellular Ca.sup.2+ (Coussin, et
al., Requirement of ryanodine receptor subtypes 1 and 2 for
Ca.sup.2+-induced Ca.sup.2+ release in vascular myocytes J. Biol.
Chem., 2000. 275(13):9596-603).
[0012] The RyR(Ca.sup.2+ spark)-BK.sub.Ca channel complex can be
viewed as a mechanism to limit smooth muscle contraction. Ca.sup.2+
spark frequency is increased when intravascular pressure is
elevated from 10 to 60 mm Hg in rat cerebral arteries (Jaggar, J.
H., Intravascular pressure regulates local and global Ca.sup.2+
signaling in cerebral artery smooth muscle cells. Am. J. Physiol.
Cell Physiol., 2001. 281(2):C439-48). Inhibition of RyR or BKCa
channels has been demonstrated to lead to pressure-induced cerebral
artery constriction (Gollasch, et al., Ontogeny of local
sarcoplasmic reticulum Ca.sup.2+ signals in cerebral arteries:
Ca.sup.2+ sparks as elementary physiological events [published
erratum appears in Circ. Res. 1999 Jan. 8-22; 84(1):125]. Circ.
Res., 1998. 83(11):1104-14; Knot, et al., Ryanodine receptors
regulate arterial diameter and wall Ca.sup.2+ in cerebral arteries
of rat via Ca.sup.2+-dependent K.sup.+ channels. J. Physiol.
(Lond), 1998. 508(Pt 1):211-21). BK.sub.Ca channel from VSMC
derived from .beta.1 subunit knockout animals demonstrated
.about.100-fold lower probability of opening and Ca.sup.2+ spark
induced BK.sub.Ca channel current was significantly reduced and
greater than 1/3 of sparks failed to elicit a BK.sub.Ca channel
activation (Brenner, et al., Vasoregulation by the .beta.1 subunit
of the calcium-activated potassium channel. Nature, 2000.
407:870-876). Mean arterial pressure was elevated in the .beta.1
subunit null animals, leading to left ventricular hypertrophy and
hypertension (Id.). Thus, the ability of the BK.sub.Ca channel to
sense the Ca.sup.2+ sparks was impaired by the loss of the .beta.1
subunit. In contrast, a gain of function mutation of .beta.1
(G352A) was associated with a low prevalence of moderate and severe
diastolic hypertension (Fernandez-Fernandez, et al.,
Gain-of-function mutation in the KCNMBI potassium channel subunit
is associated with low prevalence of diastolic hypertension. J.
Clin. Invest, 2004. 113(7): 1032-9). BK.sub.Ca-.beta.1.sub.E65K
channels showed increased Ca.sup.2+ sensitivity (Id.). Activation
of the PKA and PKG signal transduction pathways leads to 2-3 fold
increases in both Ca.sup.2+ spark and BK.sub.Ca channel activity
(Porter, et al., Frequency modulation of Ca.sup.2+ sparks is
involved in regulation of arterial diameter by cyclic nucleotides.
Am. J. Physiol, 1998. 274(5 Pt I):C1346-55; Wellman, et al., Role
of phospholamban in the modulation of arterial Ca.sup.2+ sparks and
Ca.sup.2+-activated K.sup.+ channels by cAMP. Am. J. Physiol. Cell
Physiol, 2001. 281(3):C1029-37). Ryanodine reduced dilation to
forskolin by 80%, consistent with the importance of Ca.sup.2+
sparks and a potential regulatory role of PKA. However, in arterial
smooth muscle derived from phospholamban null mice, forskolin had
little effect compared to the .about.2 fold increase in Ca.sup.2+
spark frequency in wild type animals (Wellman, et al., Role of
phospholamban in the modulation of arterial Ca.sup.2+ sparks and
Ca.sup.2+-activated K.sup.+ channels by cAMP. Am. J, Physiol. Cell
Physiol, 2001. 281(3):C1029-37).
Modulators of BK.sub.Ca Channel Activity
[0013] Acute pharmacological inhibition of BK channels has been
shown to increase ASM baseline contractility, enhance
cholinergic-mediated contraction and prevent isoproterenol-mediated
relaxation of tracheal rings (Jones et al., Selective inhibition of
relaxation of guinea-pig trachea by charybdotoxin, a potent
Ca(++)-activated K+ channel inhibitor. J Pharmacol Exp Ther
255:697-706 (1990); Murray et al., Receptor-activated calcium
influx in human airway smooth muscle cells. J Physiol 435:123-144
(1991); Corompt et al., Inhibitory effects of large
Ca.sup.2+-activated K.sup.+ channel blockers on beta-adrenergic-
and NO-donor-mediated relaxations of human and guinea-pig airway
smooth muscles. Naunyn Schmiedebergs Arch Pharmacol 357:77-86
(1998); Jones et al., Interaction of iberiotoxin with
betaadrenoceptor agonists and sodium nitroprusside on guinea pig
trachea. J Appl Physiol 74:1879-1884 (1993)).
[0014] Supporting the important role of BK channels in ASM
contractility are several recent findings. First, a report
(Sausbier et al., Reduced rather than enhanced cholinergic airway
constriction in mice with ablation of the large conductance
Ca.sup.2+-activated K.sup.+ channel. FASEB J 21:812-822 (2007))
demonstrated that the membrane potential of BK a null mice tracheal
SMC are .about.10 mV less negative than the membrane potential of
cells from WT mice. However, BK .alpha. null animals had a
paradoxical phenotype of reduced sensitivity of the airways toward
bronchoconstrictors and an enhanced sensitivity toward
bronchodilators. Both effects were the result of compensatory
mechanisms involving the amplification of cGMP signaling proteins,
suggesting that BK channels play such an important role in airway
physiology that long-term adaptation mechanisms compensate for the
loss of functional channels (Id.). Second, another report (Semenov
et al., BK channel beta1-subunit regulation of calcium handling and
constriction in tracheal smooth muscle. Am J Physiol Lung Cell Mol
Physiol 291:L802-810 (2006)) demonstrated that increased resting
[Ca.sup.2+].sub.i and increased sustained component of Ca.sup.2+
influx after cholinergic stimulation in tracheal SMC isolated from
BK .beta.1 null mice compared to WT mice. Third, in an
African-American asthmatic population, a BK .beta.1 subunit
polymorphism (R140W) is associated with a clinically significant
decline (-13%) in FEV1 in males, but not females (Seibold et al.,
An African-specific functional polymorphism in KCNMB1 shows sex
specific association with asthma severity. Hum Mol Genet
17:2681-2690 (2008)). R140W is in the extracellular loop of .beta.1
and suppresses .beta.1 enhancement of BK sensitivity to Ca.sup.2+.
It is apparent that the extracellular loop of .beta.1 plays an
important role in modulating .alpha., since a different
polymorphism in .beta.1, E65K, is associated with a decreased
incidence of diastolic hypertension and heart disease due to a
gain-of-function (Fernandez-Fernandez et at, Gain-of-function
mutation in the KCNMB1 potassium channel subunit is associated with
low prevalence of diastolic hypertension. J Clin Invest, 113(7): p.
1032-9 (2004)). Electrophysiology studies of .alpha. and R140W
mutant .beta.1 subunits demonstrated significantly reduced channel
openings.
[0015] Pharmacologic approaches to activate BK.sub.Ca channels
represent a new/emerging strategy to control membrane excitability.
Despite the increasing number of natural and synthetic BK.sub.Ca
channel openers, relatively little is known about the interaction
sites and mechanism of action. Moreover, many of the compounds are
relatively weak, with nonspecific activity towards BK.sub.Ca
channels (Ohwada, et al., Dehydroabietic acid derivatives as a
novel scaffold for large-conductance calcium-activated K.sup.+
channel openers. Bioorg. Med. Chem. Lett., 2003. 13(22):3971-4).
Small natural or synthetic products could have effectiveness in
diseases mediated through muscular and neuronal hyperexcitability
such as asthma, urinary incontinence/bladder spasm, gastroenteric
hypermotility, hypertension, coronary spasm, psychoses, convulsion
and anxiety (Calderone, V., Large-conductance, Ca.sup.2+-activated
K.sup.+ channels: function, pharmacology and drugs. Curr. Med.
Chem., 2002. 9(14):1385-95; Pelaia, et al., Potential role of
potassium channel openers in the treatment of asthma and chronic
obstructive pulmonary disease. Life Sci, 2002. 70(9):977-90;
Gribkoff, et al., The pharmacology and molecular biology of
large-conductance calcium-activated (BK) potassium channels. Adv.
Pharmacol, 1997. 37:319-48; Nardi, et at, Natural modulators of
large-conductance calcium-activated potassium channels. Planta.
Med., 2003. 69(10):885-92). Recent work has suggested a role for
K.sup.+ channel activators for post-stroke neuroprotection,
erectile dysfunction and cardiac diseases such as coronary artery
vasospasm/hypertension (Nardi, et al., Natural modulators of
large-conductance calcium-activated potassium channels. Planta.
Med., 2003. 69(10):885-92; Gribkoff, et al., Targeting acute
ischemic stroke with a calcium-sensitive opener of maxi-K potassium
channels. Nat. Med., 2001. 7(4):471-7).
[0016] The synthetic benzimidazolone derivatives NS004 and NS1619
are the pioneer BK-activators (activate BK.sub.Ca current at 10-30
.mu.M in vascular and non-vascular smooth muscle) (Coghlan, et al.,
Recent developments in the biology and medicinal chemistry of
potassium channel modulators: update from a decade of progress. J.
Med. Chem. 2001. 44(11):1627-53) and have led to the design of
several novel and heterogenous BK-openers (Olesen, et al., NS004-an
activator of Ca.sup.2+-dependent K.sup.+ channels in cerebellar
granule cells. Neuroreport, 1994. 5(8): 1001-4; Olesen, et al.,
Selective activation of Ca.sup.2+-dependent K.sup.+ channels by
novel benzimidazolone. Eur. J. Pharmacol., 1994. 251(I):53-9). In
addition to BK.sub.Ca channel opening, NS-1619 inhibits Ca.sup.2+
and Cl.sup.- channels (Gribkoff, et al., The pharmacology and
molecular biology of large-conductance calcium-activated (BK)
potassium channels. Adv. Pharmacol, 1997. 37:319-48), but has been
reported to increase intracellular Ca.sup.2+ concentration (at 30
.mu.M) in porcine coronary myocytes through the ryanodine receptor
sensitive storage sites (Yamamura, et al., BK channel activation by
NS-1619 is partially mediated by intracellular Ca.sup.2+ release in
smooth muscle cells of porcine coronary artery. Br. J. Pharmacol,
2001. 132(4):828-34). NS1608 caused BK.sub.Ca channel activation
(minimum effective concentration 0.5 .mu.M; maximum between 5-10
.mu.M), but demonstrated a bell shaped concentration with an
inhibitory effect at higher concentrations (50 .mu.M) in porcine
coronary artery cells (Hu, et al., Differential effects of the
BK.sub.Ca channel openers NS004 and NS1608 in porcine coronary
arterial cells. Eur. J. Pharmacol, 1995. 294(1):357-60; Hu, et al.,
On the mechanism of the differential effects of NS004 and NS1608 in
smooth muscle cells from guinea pig bladder. Eur. J. Pharmacol,
1996. 318:461-8). BMS-204352 (MaxiPost) has been evaluated in
clinical trials for stroke therapy and a reduction in brain infarct
size has been detected in rat stroke models (Gribkoff, et al.,
Targeting acute ischemic stroke with a calcium-sensitive opener of
maxi-K potassium channels. Nat. Med, 2001. 7(4):471-7; Imaizumi, et
al., Molecular basis of pimarane compounds as novel activators of
large-conductance Ca.sup.2+-activated K.sup.+ channel alpha-subunit
Mol. Pharmacol, 2002. 62(4):836-46). The effects of BMS-204352 were
Ca.sup.2+ sensitive; at 50 nM intracellular Ca.sup.2+, the compound
had almost no effect, whereas at higher intracellular Ca.sup.2+
concentrations, it produced progressively greater increases in
current (Gribkoff, et al., Targeting acute ischemic stroke with a
calcium-sensitive opener of maxi-K potassium channels. Nat. Med,
2001. 7(4):471-7). Three glycosylated triterpenes called
dehydrosoyasaponin-I (DHS-I), soyasaponins I and III have been
shown to activate BK.sub.Ca channels. DHS-I has poor membrane
permeability, but is probably metabolized to other active molecules
that penetrate the cell. DHS-I increases channel activity when the
.alpha. and .beta. subunits are co-expressed (McManus, et al., An
activator of calcium-dependent potassium channels isolated from a
medicinal herb. Biochemistry, 1993. 32(24):6128-33; Giangiacomo, et
al., Mechanism of maxi-K channel activation by
dehydrosoyasaponin-I. J. Gen. Physiol, 1998. 112(4):485-501).
Maxikdiol, a 1,5-dihydroxyisoprimane diterpenoid has limited
membrane permeability, but can activate the channel (threshold-1
.mu.M; significant effect-3-10 .mu.M) when applied to the
cytoplasmic side (Nardi, et al., Natural modulators of
large-conductance calcium-activated potassium channels. Planta.
Med., 2003. 69(10):885-92; Singh, et al., Maxikdiol: a novel
dihydroxyisoprimane as an agonist of maxi-K channels. J. Chem. Soc.
Perkin Trans., 1994. 1:3349-3352; Kaczorowski, et al.,
High-conductance calcium-activated potassium channels; structure,
pharmacology, and function. J. Bioenerg. Biomembr., 1996.
28(3):255-67; Lawson, K., Potassium channel openers as potential
therapeutic weapons in ion channel disease. Kidney Int., 2000.
57(3):838-45).
Rottlerin
[0017] Rottlerin (mallotoxin), a natural product from Mallotus
phillippinensis, is a
5,7-dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)-8-
-cinnamoyl-I,2-chromene that has been frequently used as a protein
kinase C.delta. (PKC.delta.) inhibitor based upon an in vitro study
demonstrating that the IC.sub.50 for PKC.delta. and CaMK III were
3-6 .mu.M compared to 30-100 .mu.M for other PKC isozymes, protein
kinase A (PKA) and casein kinase II (Gschwendt, et al., Rottlerin,
a novel protein kinase inhibitor. Biochem. Biophys. Res. Commun.,
1994. 199(I):93-8). Based upon rottlerin, a role for PKC.delta. in
a variety of biological events including apoptosis, cell
differentiation, mitogen activated protein kinase activation and
other cell processes was described. Rottlerin inhibits an increase
of histamine in BAL fluid from OVA-challenged animals compared to
animals challenged with PBS (Cho et al., "Protein kinase C.delta.
functions downstream of Ca.sup.2+ mobilization in Fc.epsilon.RI
signaling to degranulation in mast cells" J Allergy Clin Immunol,
114:1085-1092 (2004)).
[0018] More recent data suggest that rottlerin is ineffective in
blocking PKC.delta. activity in vitro, but can uncouple
mitochondria (10 .mu.M) in intact cells and reduce ATP levels in a
PKC independent fashion (Soltoff, S. P., Rottlerin is a
mitochondrial uncoupler that decreases cellular ATP levels and
indirectly blocks protein kinase C.delta. tyrosine phosphorylation.
J. Biol. Chem., 2001. 276(41):37986-92; see also Soltoff, S. P.,
Rottlerin: an inappropriate and ineffective inhibitor of
PKC.delta.. Trends in Pharm. Sci. 28(9):453-458 (August 2007)),
potentially sensitizing colon carcinoma cells to tumor necrosis
factor-related apoptosis (Tillman, et al., Rottlerin sensitizes
colon carcinoma cells to tumor necrosis factor-related
apoptosis-inducing ligand-induced apoptosis via uncoupling of the
mitochondria independent of protein kinase C. Cancer Res., 2003.
63(16):5118-25). In standard assays at 0.1 mM ATP (or even 0.01
mM), rottlerin (20 .mu.M) had virtually no effect on PKC.alpha. or
PKC.delta. activity in the presence of phosphatidylserine (PS)
using either histone H1 or myelin basic protein as a substrate
(Davies, et al., Specificity and mechanism of action of some
commonly used protein kinase inhibitors. Biochem. J., 2000. 351(R
I):95-105).
[0019] Rottlerin does inhibit several other kinases, including
p38-regulated/activated protein kinase (PRAK) and mitogen-activated
protein kinase with similar in vitro potencies as PKC.delta.. In
addition, 20 .mu.M rottlerin as been shown to substantially inhibit
c-Jun N-terminal kinase 1 .alpha.1 (JNK1 .alpha.1, 51% inhibition),
mitogen- and stress-activated protein kinase 1 (MSK-1, 62%
inhibition), PKA (83% inhibition), 3-phosphoinositide-dependent
protein kinase-1 (PDK-1, 64% inhibition), Akt (73% inhibition) and
glycogen synthase kinase 3.beta.GSK3.beta., 87% inhibition)
(Soltoff, S. P. "Rottlerin: an inappropriate and ineffective
inhibitor of PKC.delta." Trends Pharmacol Sci 28:453-458 (2007);
Davies et al., "Specificity and mechanism of action of some
commonly used protein kinase inhibitors" Biochem J 351:95-105
(2000)). Rottlerin has also been reported to inhibit
insulin-induced glucose uptake (IC.sub.50=10 .mu.M) in 3T3-L1
adipocytes (Kayali, et al., Rottlerin inhibits insulin-stimulated
glucose transport in 3T3-L1 adipocytes by uncoupling mitochondrial
oxidative phosphorylation. Endocrinology, 2002. 143(10):3834-96).
Rottlerin has been reported to decrease the capacity for the
glutamate-aspartate transporter (GLAST) subtype of the glutamate
transporter (Susarla, et al., Rottlerin, an inhibitor of protein
kinase C.delta. (PKC.delta.), inhibits astrocytic glutamate
transport activity and reduces GLAST immunoreactivity by a
mechanism that appears to be PKC.delta.-independent J. Neurochem.,
2003. 86(3):635-45).
[0020] Rottlerin also inhibits, in a dose-dependent manner,
CD4.sup.+ and CD8.sup.+ human T lymphocyte proliferation in
response to anti-CD3/anti-CD28 antibodies. The inhibition was
associated with impaired CD25 expression, decreased IL-2 production
and decreased mRNA expression of interferon .gamma., IL-10 and
IL-13 activated T cells (Springael et al., "Rottlerin inhibits
human T cell responses" Biochem Pharmacol 73:515-525 (2007)).
Rottlerin blocked PMA-induced phosphorylation of Erk-1 and Erk-2 in
Jurkat T cells and purified human CD4+ T cells from peripheral
blood (Roose et al., "A diacylglycerol-protein kinase C-RasGRP1
pathway directs Ras activation upon antigen receptor stimulation of
T cells" Mol Cell Biol 25:4426-4441 (2005)).
[0021] The history/development of rottlerin as a therapeutic
compound is complex. Kamala, from which rottlerin may be purified,
has been used in India for centuries as an antihelmintic. Kamala is
collected from the capsules of Mallotus philippinensis, a tree
grown in Abyssinia, Southern Arabia, Hindostan, the East India
Islands, China, and Australia (Remington et al., ed, The
Dispensatory of the United States of America, 1918). Kamala, when
examined under the microscope, consists of garnet-red,
semi-transparent, roundish, glandular hairs from 0.040 to 0.100 mm
in diameter, and containing numerous red, club-shaped cells and
admixed with minute stellate hairs, and the remains of stalks and
leaves, the latter of which are easily removed by careful sifting.
(Id.) The most important constituent of Kalama is a dark brownish
red resin (about 80%) composed chiefly of a crystalline chemical,
rottlerin and a yellowish crystalline isomer, isorottlerin.
(Gujral, et al., Oral contraceptives. Part II. Antifertility effect
of Mallotus philippinensis Mueller-argoviensis. Indian J. Med.
Res., 1960. 48:52-8). Thomas Anderson of Glasgow found that kamala
consists of 78.19% resinous coloring matter, 7.34% albumin, 7.14%
cellulose and the like, a trace of volatile oil and volatile
coloring matters, 3.84% ashes, and 3.49% water. (Remington et al.,
ed, The Dispensatory of the United States of America, 1918) The
amount of earthy impurities, chiefly sand, in commercial kamala,
varies greatly, sometimes amounting to fifty or even sixty per
cent. (Id.)
[0022] Kamala is violently purgative in full doses, occasionally
causing nausea but seldom vomiting (Gujral, et al., Oral
contraceptives. Part II. Antifertility effect of Mallotus
philippinensis Mueller-argoviensis. Indian J. Med. Res., 1960.
48:52-8). In 1910, Semper reported that kamala caused a paralyzing
effect on motor nerves and muscle (Id.), which based on the
inventors' data is likely due to its effects on the BK.sub.Ca
channel. Kamala has been used in India against the tapeworm and is
given to patients (3.9-11.6 grams) suspended in water, mucilage or
syrup. The worm is usually expelled at the third or fourth stool.
As an external remedy, kamala is used by the people of India for
various afflictions of the skin, particularly scabies.
[0023] Rottlerin appears to have been isolated .about.150 years ago
(1855) by Anderson (Anderson, A., Kamala resin-rottlerin. Edin. New
Phil. Jour., 1855. 1:296-300), by allowing a concentrated ethereal
solution of kamala to stand for two days, draining and pressing the
granular crystals in bibulous paper and purifying them from the
adhering resin. Perkin and Perkin confirmed the substance and
called it mallotoxin (Gujral, et al., Oral contraceptives. Part II.
Antifertility effect of Mallotus philippinensis
Mueller-argoviensis. Indian J. Med. Res., 1960. 48:52/-8). Such
isolation methods are incorporated by reference as if recited in
full herein. Pure rottlerin has the chemical composition
C.sub.33H.sub.30O.sub.9 and has been reported to exist in both the
keto and enol forms.
[0024] Rottlerin has been given to animals; it has been shown to
reduce the fertility rate of rats and guinea pigs (dose of purified
rottlerin=10-20 mg/kg/day.times.6 days) (Id.). Injection of
rottlerin (5 .mu.M) into the cistera magna in a canine subarachnoid
hemorrhage model inhibited the initial phase of cerebral vasospasm,
which was attributed to its effects on PKC.delta. (Nishizawa, et
al., Attenuation of canine cerebral vasospasm after subarachnoid
hemorrhage by protein kinase C inhibitors despite augmented
phosphorylation of myosin light chain. J. Vase. Res., 2003.
40(2):169-78). It is conceivable that some of the beneficial
effects may have been secondary to the effects on BK.sub.Ca
channel. The RTECS database (AM6913800) indicates no information
regarding LD.sub.50/LC.sub.50 for acute/chronic toxicity.
[0025] In view of the foregoing, new and improved methods and
compositions for modulating ASM would be desirable. The present
invention is directed to achieving these and other objectives.
SUMMARY OF THE INVENTION
[0026] Disclosed herein is that rottlerin and derivatives thereof
are potent activators of the BK channel and that asthma,
hypertension, and related disorders can be treated or prevented via
regulation of the BK channel using rottlerin. Accordingly, the
present invention provides compositions and methods for regulating
the BK channel using rottlerin and derivatives thereof.
Pharmaceutical compositions and methods for treating, preventing,
or ameliorating the effects of asthma are also provided.
[0027] One embodiment of the present invention is a method of
treating or ameliorating the effects of a disease characterized by
altered smooth muscle contractility. This method comprises
administering to a patient suffering from such a disease an
effective amount of a large-conductance Ca.sup.2+-activated K.sup.+
(BK) channel modulator.
[0028] Another embodiment of the present invention is a method of
treating or ameliorating the effects of asthma. This method
comprises administering to a patient suffering from asthma an
effective amount of a BK channel modulator.
[0029] A further embodiment of the present invention is a method
for decreasing airway constriction and/or airway resistance in a
patient without increasing the heart rate of the patient. This
method comprises administering to the patient an effective amount
of a BK channel modulator or a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and a BK channel
modulator.
[0030] Yet another embodiment of the present invention is a method
for modulating inflammation in a lung of a patient. This method
comprises administering to a patient an effective of amount of a BK
channel modulator, or a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and a BK channel modulator,
which amount is sufficient to modulate the inflammation.
[0031] An additional embodiment of the present invention is a
pharmaceutical composition for treating or ameliorating the effects
of a disease characterized by altered smooth muscle contractility.
This pharmaceutical composition comprises a pharmaceutically
acceptable carrier and a BK channel modulator.
[0032] Another embodiment of the present invention is a
pharmaceutical composition for treating, preventing, or
ameliorating the effects of asthma. This pharmaceutical composition
comprises a pharmaceutically acceptable carrier and a BK channel
modulator.
[0033] In another aspect of the present invention, the
above-described compounds and pharmaceutical compositions can be
used to regulate membrane excitability both in vitro and in vivo.
In one example, the compounds and pharmaceutical compositions of
the present invention can be used to treat or prevent a
hyperexcitability disorder.
[0034] In an embodiment of the invention, the hyperexcitability
disorder is asthma. In another embodiment of the invention, the
hyperexcitability disorder is hypertension. In other embodiments of
the present invention, the hyperexcitability disorder includes, but
is not necessarily limited to urinary incontinence, gastroenteric
hypermotility, coronary spasm, psychoses, convulsion and anxiety.
In another embodiment, the compounds and pharmaceutical
compositions of the present invention are used in treating or
preventing erectile dysfunction. In yet other embodiments, the
compounds and pharmaceutical compositions of the present invention
are used in treating or preventing coronary artery vasospasm and
hypertension. In another embodiment, the compounds and
pharmaceutical compositions of the present invention are used in
treating or preventing neurologic dysfunction. In an additional
embodiment, the compounds and pharmaceutical compositions of the
present invention are used in post-stroke neuroprotection.
[0035] The present invention also provides methods for treating or
preventing a hyperexcitability disorder in a subject, comprising
administering to the subject a therapeutically effective amount of
the pharmaceutical composition of the invention. In an embodiment
of the invention, the hyperexcitability disorder includes, but is
not necessarily limited to, asthma, urinary incontinence,
gastroenteric hypermotility, hypertension, coronary spasm,
psychoses, convulsion and anxiety.
[0036] The present invention also provides methods for treating or
preventing erectile dysfunction in a subject by administering to
the subject a therapeutically effective amount of a pharmaceutical
composition of the invention. Additionally, the present invention
also provides methods for treating or preventing a coronary artery
vasospasm in a subject, comprising administering to the subject a
therapeutically effective amount of a pharmaceutical composition of
the present invention.
[0037] The invention additionally provides methods for treating or
preventing hypertension in a subject, comprising administering to
the subject a therapeutically effective amount of a pharmaceutical
composition of the present invention.
[0038] The present invention further encompasses methods for
treating or preventing a neurologic dysfunction in a subject,
comprising administering to the subject a therapeutically effective
amount of a pharmaceutical composition of the present invention.
The present invention also provides methods for post-stroke
neuroprotection in a subject by administering a therapeutically
effective amount of a pharmaceutical composition of the present
invention.
[0039] The present invention further provides kits for use in
treating or preventing hyperexcitability disorders in a subject
comprising a therapeutically effective amount of a pharmaceutical
composition of the present invention, optionally, in combination
with a pharmaceutically acceptable carrier. In an embodiment, the
hyperexcitability disorder includes, but is not necessarily limited
to, asthma, urinary incontinence, gastroenteric hypermotility,
hypertension, coronary spasm, psychoses, convulsion and
anxiety.
[0040] The present invention also provides kits for use in treating
or preventing erectile dysfunction, coronary artery vasospasm,
hypertension or neurologic dysfunction in a subject, comprising
administering a therapeutically effective amount of a
pharmaceutical composition of the present invention.
[0041] Finally, the present invention also provides kits for use in
post-stroke neuroprotection in a subject, comprising a
therapeutically effective amount of a pharmaceutical composition of
the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0042] FIG. 1 depicts a representation of PKA and .beta.1-subunit
regulation of BK.sub.Ca channel. FIG. 1A illustrates that signaling
through the BK.sub.Ca-associated .beta.2AR leads to cAMP
generation, PKA phosphorylation of S872 (mSlo), and increased
channel activity. Increased channel activity may be due to G.alpha.
association. FIG. 1B illustrates that .beta.1 subunit modification
of BK.sub.Ca channel leads to activation.
[0043] FIG. 2 shows a schematic of the structure of an .alpha.
subunit of the BK.sub.Ca channel. The .alpha. subunit is the pore
forming subunit; the tetrameric channel is formed by four .alpha.
subunits. Seven transmembrane domains are shown: S0-S6. The pore is
between S5 and S6. The channel has a unique C-terminus, with four
additional, non-transmembrane hydrophobic regions (S7-S10). The
Ca.sup.2+ regulatory domains are indicated; the Ca.sup.2+ bowl,
M513 and D362/D367 form independent high affinity Ca.sup.2+
sensors. The RCK1 and RCK2 domains are indicated. Adapted from
(Magleby, K. L., Gating mechanism of BK (Slo1) channels: so near,
yet so far. J. Gen. Physiol, 2003. 121(2):81-96).
[0044] FIG. 3 is a schematic representation of the role of RyR in
regulation of smooth muscle cell (SMC) constriction and dilation.
Local Ca.sup.2+ release (sparks) from RyR activate BK.sub.Ca, whose
outward current (spontaneous transient outward currents; STOC)
hyperpolarize the membrane and inhibit voltage gated Ca.sup.2+
channels (Ca.sub.v1.2). Agents that increase cAMP in vascular SMC
cause vasodilatation. PKA has a direct effect on BK.sub.Ca, but
also increases spark activity, potentially by increased
phosphorylation of the voltage gated Ca.sup.2+ channel, RyR, and
phospholamban (adapted from (Porter, et al., Frequency modulation
of Ca.sup.2+ sparks is involved in regulation of arterial diameter
by cyclic nucleotides. Am. J. Physiol., 1998. 274 (Cell Physiol.
43):C1346-C1355)). The presence of the IP3R on the sarcoplasmic
reticulum (SR) is not shown. The .beta.2AR is shown associated with
BK.sub.Ca and Ca.sub.v1.2, whereas .beta.1 adrenergic receptor (AR)
does not associate with either channel. The regulation of BK.sub.Ca
and RyR by other kinases is not shown.
[0045] FIG. 4 shows electrophysiologic characterization of
rottlerin. FIGS. 4A and 4B show representative current traces from
whole cell patches with 5 mM EGTA in patch pipette. Rottlerin (0.5
.mu.M) was applied to the extracellular side through local
perfusion. Voltage steps are shown at the right of each tracing;
note the different maximum voltage steps +200 mV (upper) vs. +120
mV (lower). Rottlerin significantly prolonged tail currents
indicative of slowing of deactivation. Tail currents are in the
opposite direction due to the final voltage step (+60 upper, -60 mV
left). FIG. 4C G-V curves were constructed for indicated conditions
utilizing tail analysis (Xia, et al., Multiple regulatory sites in
large-conductance calcium-activated potassium channels. Nature,
2002. 418(6900):880-4). Solid lines were fitted with Boltzmann
function. Exposure to rottlerin shifted the G-V curve in the
hyperpolarizing direction (indicative of activation).
Representative of more than 30 similar experiments with rat and
murine (pictured) slo. FIGS. 4D and 4E show experiments studying
stable mslo HEK293 cells in whole cell configuration with a pipette
solution containing 5 mM EGTA (.about.0 free Ca.sup.2+). Local
perfusion of rottlerin (extracellular; 0.5 .mu.M) activated
channel. Exposure of cell to BK.sub.Ca channel blocker
tetraethylammonium (TEA) (5 mM) reversibly inhibited rottlerin
induced BK.sub.Ca activation.
[0046] FIG. 5 shows that intracellular exposure of rottlerin
through dialysis for an extended period has minimal effect on
BK.sub.Ca channel activity. FIG. 5A shows representative current
traces made 1 minute and 25 minutes after establishing whole cell
voltage patch clamp, dialyzed with 5 mM EGTA (.about.0 cytosolic
Ca.sup.2+) and 20 .mu.M rottlerin in a patch pipette. Over 25
minutes, intracellular dialysis of rottlerin had minimal effect on
BK.sub.Ca current. After 25 minutes, the cell was exposed to
rottlerin (0.5 .mu.M) via local perfusion, which significantly
increased channel activity as shown in the diary plot (FIG. 5B) and
the G-V curve (FIG. 5C). Current in the diary plot in FIG. 5B
represents maximal current in ramp protocol at +70 mV. Holding
potential of ramp was -20 mV, with ramp from -30 mV to +180 mV over
500 ms. The G-V curve in FIG. 5C was generated from the tail
analysis as described in FIG. 4. Exposure of rottlerin through
cytoplasmic administration had minimal effect on the channel; only
exposure through the extracellular space caused activation,
suggesting that the compound requires access to the privileged
space only accessible extracellularly.
[0047] FIG. 6 shows single channel recordings of a BK.sub.Ca
channel. FIG. 6A shows representative outside-out single channel
traces demonstrating activation of a BK.sub.Ca channel from local
perfusion of rottlerin (0.5 .mu.M) to the extracellular side.
Amplitude histograms are shown on the right. Rottlerin does not
significantly change the channel conductance. Ca.sup.2+ was
maintained at 0 (virtual; actual <20 nM) through dialysis using
a patch pipette. Because cellular compartments are excluded from
patch and outside-out patch perfused locally with 0 Ca.sup.2+,
activation is Ca.sup.2+-independent. Channel activation can be
inhibited by iberiotoxin or TEA (not shown). FIG. 6B shows
representative inside-out single channel traces demonstrating
activation of BK.sub.Ca with local perfusion of rottlerin (0.5
.mu.M) in the presence of 0 Ca.sup.2+. Amplitude histograms are
shown on the right.
[0048] FIG. 7 shows that rottlerin activates BK.sub.Ca channel in
Human Embryonic Kidney (HEK) and VSMC cells. (A) Comparison of the
effects of NS-1619 and rottlerin. Time course of whole cell voltage
clamp experiment in a stably transfected mSlo HEK293 cell
demonstrating current at +60 mV. Current was monitored with a ramp
protocol; holding potential -60 mV, with ramp from -80 mV to +150
mV over 500 ms. Extracellular application of NS-1619 (10 .mu.M)
increased current as previously described (Olesen, et al.,
Selective activation of Ca.sup.2+-dependent K.sup.+ channels by
novel benzimidazolone. Eur. J. Pharmacol., 1994. 251(1):53-9).
After stabilization of the current, rottlerin (0.5 .mu.M) was
applied to the cell by local perfusion. Rottlerin shifted the
V.sub.0.5 by .about.100 mV after 5 minutes. Analysis was performed
using tail analysis with normalization as previously described
(Xia, et al., Multiple regulatory sites in large-conductance
calcium-activated potassium channels. Nature, 2002.
418(6900):880-4). FIG. 7B shows a study using HEK cells
co-expressing .alpha. and .beta.1 subunits in whole cell,
configuration with 5 mM EGTA (intra-pipette). Rottlerin (0.5 .mu.M)
significantly shifted the I-V curve to the left, similar to results
in HEK cells expressing only the .alpha. subunit (see FIG. 5). FIG.
7C shows a study using human VSMC in outside-out configuration,
utilizing single channel recordings, recorded at +60 mV. Exposure
of the same patch to rottlerin (0.5 .mu.M) by local perfusion
significantly increased Po and open dwell time. Identification of
the BK.sub.Ca channel was determined by conductance and inhibition
by iberiotoxin and TEA. FIG. 7D shows that rottlerin inhibits
phenylephrine (PE) induced modulation of VSMC tone. Murine femoral
arterial rings were isolated and placed in a wire myograph. PE
induced constriction of the vessel was significantly inhibited by
rottlerin (0.5 .mu.M). Rottlerin's effects were inhibited by TEA.
The figures shown are representative of 4 similar experiments.
Error bars are standard error of the mean (SEM). Asterisk (*)
indicates p<0.001.
[0049] FIG. 8 shows the results of tracheal constriction studies.
FIG. 8A shows the cumulative dose-response curves of WT tracheal
rings to isoproterenol in the presence of a .beta.1-AR antagonist
(CGP 20712A; 100 nM) or a .beta.2-AR antagonist (ICI 118551; 100
nM) or vehicle (DMSO). (n=4 each). Asterisk (*) indicates P<0.05
versus vehicle (DMSO). FIG. 8B shows that BK channels are required
for .beta.-agonist mediated tracheal ring relaxation. Cumulative
dose-response curves of WT tracheal rings to isoproterenol in the
absence and presence of 100 nM iberiotoxin (IbTX), a specific BK
channel inhibitor. Values in graphs are means.+-.standard
deviation.
[0050] FIG. 9A shows the timeline of rottlerin administration in an
ovalbumin (OVA) induced acute asthma model. FIG. 9B shows airway
responsiveness in mice following OVA challenge and rottlerin
administration. n=4 per group. Asterisk (*) indicates P<0.05 for
OVA compared to OVA+Rottlerin.
[0051] FIG. 10 shows that rottlerin reduces the inflammatory
response in asthma. FIG. 10A shows differential cell count of
bronchoalveolar lavage fluid (BALF) from lungs of mice. Data are
expressed as mean+SEM. (n=17 per group). **p<0.01 (OVA+PBS
compared to PBS+PBS); *p<0.05 (OVA+PBS compared to
OVA+rottlerin); #p<0.05, ##p<0.01 (OVA+rottlerin compared to
PBS+Rottlerin). FIG. 10B shows the levels of OVA specific IgE as
determined by ELISA. Data are expressed as mean+S.D. (n=17 per
group). **p<0.01 (OVA+PBS compared to PBS+PBS); ##p<0.01
OVA+rottlerin compared to PBS+rottlerin. FIG. 10C shows the levels
of cytokines in BALF. BALF from 3-week, HDM-sensitized mice treated
with PBS or rottlerin analyzed for Th2 cytokines IL-4, IL-5 and
IL-13 (n=17 per group). *p<0.05 OVA+PBS compared to
OVA+rottlerin; ##p<0.01 OVA+PBS/rottlerin compared to
PBS+PBS/rottlerin.
[0052] FIG. 11 shows that rottlerin activates BK channels and
hyperpolarizes the membrane potential. FIG. 11 shows G-V curves,
which were generated from tail current analysis for control
conditions (triangle) and after rottlerin (0.5 .mu.M) exposure
(squares) utilizing Boltzmann function. In this experiment, murine
tracheal smooth muscle cells were acutely isolated and whole cell
patch clamped as previously described (Zakharov, S. et al., (2005)
J Biol Chem 280, 30882-30887). FIG. 11B shows representative trace
of membrane potential measurement before and after application of
rottlerin (1 .mu.M) and paxilline (10 .mu.M). FIG. 11D shows a
graph of membrane potential changes in ASM following rottlerin and
paxilline administration. FIG. 11C shows cumulative dose-response
curves of WT tracheal rings to ISO (1 nM to 100 .mu.M) in the
presence of PBS, rottlerin or rottlerin+iberiotoxin (n=5 per
group). Values are means+S.D. *p<0.05 for rottlerin compared to
rottlerin+iberiotoxin.
[0053] FIG. 12 shows that rottlerin activation of BK channels is
not dependent on cellular signaling pathways. FIG. 12A shows
representative outside-out patch single channel traces recorded at
+30 mV of recombinant BK (mSlo1) from control conditions and after
bath application of rottlerin (0.5 .mu.M). Activation was found in
100% of experiments (n=18). FIG. 12B shows representative
outside-out patch single channel traces of cultured human VSMC,
recorded at +60 mV in .about.20 nM Ca.sup.2+, from control
conditions and after bath application of rottlerin (0.5 .mu.M).
[0054] FIG. 13 shows that rottlerin enhances the
isoproterenol-induced relaxation of tracheal rings on a myograph.
Cumulative dose-response curves of WT tracheal rings of PBS- &
OVA-sensitized groups to isoproterenol with or without rottlerin
(n=5 per group) are shown. Values are means.+-.S.D. Asterisk (*)
indicates P<0.05 for OVA compared to OVA+Rottlerin.
[0055] FIG. 14 shows that rottlerin reduces airway resistance in an
Ova-sensitized asthma model. FIG. 14A shows airway responsiveness
in mice following OVA challenge and rottlerin administration. The
number of mice in each group is as follows: PBS/PBS n=4,
PBS/Rottlerin n=5, Ova/PBS n=6, Ova/Rottlerin n=5. Triple asterisks
(***) indicate p<0.001 and asterisk (*) indicates p<0.05 for
OVA/PBS compared to OVA/Rottlerin. FIG. 14B shows airway
responsiveness in mice following OVA challenge and rottlerin
administration in response to isoproterenol (n=4 per group).
Asterisk (*) indicates P<0.05 for OVA compared to
OVA+Rottlerin.
[0056] FIG. 15 shows that rottlerin activates airway smooth muscle
BK channels. The figure shown is representative of 3 similar
experiments in which tracheal smooth muscle cells were acutely
isolated from mice and exposed to rottlerin (2 .mu.M), and the
membrane potential was determined using perforated patch.
[0057] FIG. 16 shows experiments using an OVA-induction of murine
asthma model. FIG. 16A shows the protocol for asthma induction.
FIG. 16B shows pulmonary resistance (R.sub.L) as measured in
tracheostomized, and ventilated mice. RL is an indicator for airway
hyperresponsiveness. FIG. 16C shows BAL cells post antigen
sensitization and challenge in comparison with control mice.
[0058] FIG. 17 shows the inflammatory response in control and
OVA-sensitized asthma model. FIG. 17 shows hematoxylin and eosin (H
& E) stain of lungs from PBS- and OVA-sensitized animals. Lungs
were stained with H & E stain and imaged under low power
(4.times.). Note peribronchial and perivascular cellular
infiltrates in OVA-sensitized animals. Rottlerin-treated,
OVA-sensitized/challenged animals demonstrate marked reduction in
cellular infiltrates. Images are representative of results from 5-6
animals for each experimental condition.
[0059] FIG. 18 shows that a single dose of rottlerin causes
reduction in airway resistance in the OVA-asthma model. The
experimental conditions of the results shown in FIG. 18A are as
follows. Rottlerin (5 .mu.g/g) or PBS was given via the tail vein
of mice 5 minutes prior to airway resistance measurements in
OVA-challenged/sensitized animals (OVA) and
non-sensitized/challenged animals (control). N=8 in each group; *,
p<0.05; OVA; rottlerin-treated compared to OVA; PBS-treated. The
experimental conditions of the results shown in FIG. 18B are as
follows. PBS, Isoproterenol (2.5 .mu.g/g) or Isoproterenol (2.5
.mu.g/g)+Rottlerin (5 .mu.g/g) were given via the tail vein as
above.
[0060] FIG. 19 shows that rottlerin reduces airway resistance in a
house dust mite (HDM) sensitized asthma model. FIG. 19A shows the
protocol for asthma induction using the HDM model. The * represents
the days when rottlerin was administered I.P during the course of
asthma induction. FIG. 19B shows airway hyperresponsiveness (AHR)
in mice following HDM challenge and rottlerin administration. (n=4
per group). *p<0.05; HDM/PBS compared to HDM/rottlerin.
[0061] FIG. 20 shows that rottlerin inhibits inflammatory response
in HDM-exposed mice. H & E stain of lungs from PBS and
HDM-exposed animals are shown. Lungs were stained with H & E
stain and imaged under low power (4.times.). Images are
representative of similar results from 4 animals for each
experimental condition. Note peribronchial and perivascular
cellular infiltrates in HDM-exposed animals. Rottlerin-treated,
HDM-exposed animals demonstrated marked reduction in cellular
infiltrates.
[0062] FIG. 21 shows cumulative dose-response curves of WT tracheal
rings of OVA-sensitized groups to isoproterenol (ISO) (1 nM to 100
.mu.M) in the presence of PBS, rottlerin or rottlerin+iberiotoxin
(IbTX) (n=5 per group). Values are means.+-.S.D. *p<0.05 for PBS
compared to rottlerin.
[0063] FIG. 22A shows the protocol for an OVA-induced asthma model.
Groups of mice received an I.P. injection of OVA/Alum complex on
days 0 and 7 and on alternate days 14-22, a 20 minute aerosol
challenge of either PBS or 2% (w/v) OVA in PBS, using an ultrasonic
nebulizer. FIG. 22B shows that the asthma model exhibited an
increase in AHR as shown by an increase in R.sub.L in response to
MCh.
[0064] FIG. 23 shows the ISO-induced increase in outward K.sup.+
currents in acutely isolated tracheal smooth muscle. FIG. 23A is a
diary plot of current recorded during repetitive stimulation by
depolarizing ramps every 5 seconds to +200 mV from a holding
potential of -20 mV. ISO and ISO+IbTX exposure are indicated by
bars at bottom of plot. Dashed line indicates average control
current. FIG. 23B shows I-V curves for control, ISO (0.5 mM) and
ISO+IbTX (100 nM). Insets demonstrate a series of current traces
for voltage steps from a holding potential of -80 mV, with steps
from +10 to +220 mV.
[0065] FIG. 24 shows the electrophysiological characterization of
selected rottlerin derivatives. Two derivatives of rottlerin are
shown, methylated rottlerin and reduced rottlerin. FIG. 24A-C show
the time course of onset (ON) of the effect of rottlerin or its
derivatives (0.5 .mu.M for rottlerin, 1 .mu.M for methylated and
reduced rottlerin) and washout (WASH). Electrophysiology was
performed using whole-cell patch clamp with a ramp every 5 seconds.
FIG. 24D-F show the current traces (insets) from whole cell voltage
clamp recordings ([Ca.sup.2+].sub.i .about.20 nM for FIG. 24D, 1
.mu.M for FIG. 23E-F) from HEK cells stably expressing BK channels
under control conditions and after rottlerin bath application. In
FIG. 24F, .DELTA.=wash. G-V curves were generated from tail current
analysis for control conditions and after rottlerin or
rottlerin-derivative exposure utilizing Boltzmann function.
DETAILED DESCRIPTION OF THE INVENTION
[0066] One embodiment of the present invention is a method of
treating or ameliorating the effects of a disease characterized by
altered smooth muscle contractility. This method comprises
administering to a patient suffering from such a disease an
effective amount of a large-conductance Ca.sup.2+-activated K.sup.+
(BK) channel modulator.
[0067] As used herein, in relation to a disease, the term
"characterized by" means one of the characteristics or one of the
symptoms of the disease. The term "altered" means different from
the norm (i.e. the population at large or an individual not
suffering from such a disease). The term "smooth muscle" refers to
a group of non-striated muscles, generally found in the walls of
the hollow organs of the body (except the heart), including but not
limited to the blood vessels, the respiratory tract, the
gastrointestinal tract, the bladder, or the uterus. The term
"contractility" refers to properties associated with the
contraction (e.g., of smooth muscle), such as contraction and
relaxation of smooth muscles. The contraction and relaxation of
smooth muscles is usually not under voluntary control.
[0068] As used herein, a "large-conductance Ca.sup.2+-activated
K.sup.+ (BK) channel" means an ion channel that conducts potassium
(K.sup.+) ions through cell membranes, and that upon opening or
activation, causes transient membrane hyperpolarization, inhibition
of Ca.sup.2+ influx through voltage-dependent Ca.sup.2+ channels,
reduced intracellular concentration of Ca.sup.2+ and smooth muscle
relaxation. A BK channel modulator is a substance that changes the
activity or the opening or the closing of the BK channel.
Preferably, the BK channel modulator is a BK channel activator. As
used herein, "a BK channel activator" means a substance, such as,
e.g., all molecules having rottlerin-type activity, that opens the
BK channels. BK channel activators may be selected from the group
consisting of rottlerin, flindokalner (Bristol-Myers Squibb),
BMS-554216 (Bristol-Myers Squibb), Pharmaprojects No. 4420 (Merck
& Co) (disclosed in U.S. patent application Ser. No. 09/516,442
filed Dec. 13, 1993), Pharmaprojects No. 4494 (Merck & Co)
(disclosed in U.S. patent application Ser. No. 09/519,771 filed
Jan. 24, 1994), NS-1619 (NeuroSearch), NSD-551 (NeuroSearch), NS-8
(Nippon Shinyaku), a pharmaceutically acceptable salt thereof, and
combinations thereof. Preferably, the BK channel activator is
rottlerin.
[0069] In the present invention, "rottlerin" is preferably used in
an isolated or purified form, either in its keto or enol form. The
purified form may be a purified extract from a natural source or a
purified compound, which is synthesized. As used herein, "isolated"
means that the rottlerin is separated from other components of
either (a) a natural source, such as a plant, as disclosed
previously herein or (b) a synthetic organic chemical reaction
mixture, suitably, via conventional techniques, wherein the
rottlerin of the invention is purified. As used herein, "purified"
means that when isolated, the isolate contains at least about 20%,
including 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% of
rottlerin by weight (wt %) of the isolate. Highly purified
rottlerin are also contemplated, wherein the isolate contains at
least 80%, preferably at least 90%, such as at least 91, 92, 93,
94, 95, 96, 97, 98, 99 or 100% of rottlerin by weight (wt %) of the
isolate. In the present invention, isolate or isolated in regards
to rottlerin includes extracts from the native plant, Mallotus
phillippinensis (e.g., red kamala powder). Rottlerin may be
isolated using methods well-known in the art, such as those
published by Anderson and Robertson et al. (Anderson, A. "Kamala
resin-rottlerin," Edin. New Phil. Jour., Vol. 1, pp. 296-300
(1855); Robertson et al., "Rottlerin." J. Chem. Soc., Part I, pp.
1862-1865 (1937)). Such methods are incorporated by reference as if
recited in full herein.
[0070] In the present invention, an "effective amount" or
"therapeutically effective amount" of a BK channel modulator is an
amount of such BK channel modulator that is sufficient to effect
beneficial or desired results as described herein when administered
to a patient, which is a mammal, preferably a human. A BK channel
modulator may also be administered as part of a pharmaceutical
composition, such as in a unit dosage form. Preferably, such a unit
dosage form is inhaled.
[0071] Furthermore, the pharmaceutical composition may be
co-administered. In the present invention, "co-administration"
includes administration of a pharmaceutical composition comprising
a BK channel modulator along with another compound, composition, or
pharmaceutical composition together in the same composition,
simultaneously in separate compositions, or as separate
compositions administered at different times, as deemed most
appropriate by a physician.
[0072] When a BK channel modulator is co-administered with another
compound or composition, that compound or composition is preferably
a conventional drug for modulating constriction of ASM such as e.g.
corticosteroids, anti-cholinergics, anti-leukotrienes,
.beta.-agonists, and/or phosphodiesterase inhibitors. Preferably,
the BK channel modulator is co-administered with a .beta.-agonist.
Non-limiting examples of a corticosteroid according the present
invention include cromolyn sodium, nedocromil, fluticasone,
budesonide, triamcinolone, flunisolide, and beclomethasone. A
non-limiting example of an anti-cholinergic according the present
invention includes ipratropium bromide. Non-limiting examples of an
anti-leukotriene according the present invention include
montelukast, zafirlukast, and zileuton. Non-limiting examples of a
.beta.-agonist according the present invention include albuterol,
levalbuterol, salmeterol, formoterol, isoproterenol, and
pirbuterol. Non-limiting examples of a phosphodiesterase inhibitor
according the present invention include ibudilast, theophylline,
CDP840, roflumilast, cilomilast,
4-(3-butoxy-4-methoxyphenyl)methyl-2-imidazolidone (Ro 20-1724),
(R)--N-(4-[1-3-cyclopentyloxy-4-methoxyphenyl)-2-(4-pyridyl)eth-
yl]phenyl)-N'-ethylurea (CT-2450),
6-(4-pyridylmethyl)-8-(3-nitrophenyl)quinoline (PMNPQ), R-rolipram,
oglemilast (Glenmark Pharmaceuticals), IPL512602 (Inflazyme
pharmaceuticals),
N-(3,5-dichloropyrid-4-yl)-[1-(4-fluorobenzyl)-5-hydroxy-indole-3-yl]-gly-
oxylic acid amide (AWD 12-281), and UK-500001 (Spina, D., PDE4
inhibitors: current status, British J. Pharmacology, 2008.
155:308-315). Co-administration of a BK channel modulator and such
drugs leads to synergism (i.e., greater than additive effects). In
view of this, lower doses of such drugs may be used in conjunction
with a BK channel modulator, which may result in lower overall side
effects.
[0073] Effective dosage forms, modes of administration, and dosage
amounts of, e.g., a BK channel modulator, may be determined
empirically, and making such determinations is within the skill of
the art. It is understood by those skilled in the art that the
dosage amount of, e.g., a BK channel modulator, will vary with the
route of administration, the rate of excretion, the duration of the
treatment, the identity of any other drugs being administered, the
age, size, and species of mammal, e.g., human patient, and like
factors well known in the arts of medicine and veterinary medicine.
In general, a suitable dose of a rottlerin (or a pharmaceutically
acceptable salt thereof) according to the invention will be that
amount of the rottlerin (or the pharmaceutically acceptable salt
thereof), which is the lowest dose effective to produce the desired
effect with no or minimal side effects.
[0074] A suitable, non-limiting example of a dosage of a BK channel
modulator according to the present invention is from about 10 ng/kg
to about 1000 mg/kg, such as from about 1 mg/kg to about 100 mg/kg,
including from about 5 mg/kg to about 50 mg/kg, about 1 mg/kg to
about 10 mg/kg, about 1 mg/kg to about 3 mg/kg, or about 5 mg/kg to
about 7 mg/kg. Other representative dosages of a BK channel
modulator include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20
mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg,
60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150
mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500
mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg.
The effective dose of a BK channel modulator maybe administered as
two, three, four, five, six or more sub-doses, administered
separately at appropriate intervals throughout the day.
[0075] In one aspect of this embodiment, diseases characterized by
altered smooth muscle contractility include e.g., pneumoconiosis
(such as aluminosis, anthracosis, asbestosis, chalicosis, ptilosis,
siderosis, silicosis, tabacosis, berylliosis, and byssinosis),
chronic obstructive pulmonary disease (COPD), asthma, bronchitis,
exacerbation of airway hyperreactivity or cystic fibrosis, cough
(including chronic cough), other pulmonary diseases, including
other reversible airway diseases, urinary incontinence, and
hypertension. Preferably, the disease is asthma, chronic
obstructive pulmonary disease, urinary incontinence, or
hypertension. More preferably, the disease is asthma.
[0076] BK channel modulators disclosed herein may be used to treat
acute or chronic diseases according to the methods disclosed
herein. As used herein, an "acute" disease means a disease with a
rapid onset (i.e., less than 5 minutes) of the symptoms, which may
have a dramatic effect on the patient. A non-limiting example of an
acute disease is an acute asthma attack, in which the individual
may have breathing difficulties and even lose consciousness in an
instant. A "chronic" disease means a long-lasting disease or
recurrent disease. Chronic asthma is one of many examples of such
chronic diseases.
[0077] Another embodiment of the present invention is a method of
treating or ameliorating the effects of asthma. This method
comprises administering to a patient suffering from asthma an
effective amount of a BK channel modulator.
[0078] A BK channel modulator may also be administered as part of a
pharmaceutical composition, such as in a unit dosage form.
Preferably, such a unit dosage form is inhaled. Furthermore, the
pharmaceutical composition may be co-administered as described
above. Preferably, the BK channel modulator is co-administered with
a .beta.-agonist.
[0079] An additional embodiment of the present invention is a
method for decreasing airway constriction and/or airway resistance
in a patient without increasing the heart rate of the patient or
with no or decreased side effects normally associated with
conventional therapy, e.g., tachycardia when .beta..sub.2 agonists
are used. This method comprises administering to the patient an
effective of amount of a BK channel modulator or a pharmaceutical
composition comprising a pharmaceutically acceptable carrier and a
BK channel modulator.
[0080] As used herein, "airway constriction" means narrowing of air
passages of the lungs, such as from smooth muscle contraction.
"Airway resistance" means obstruction to airflow provided by the
conducting airways, such as, those found in obstructive lung
diseases.
[0081] In one aspect of this embodiment, the pharmaceutical
composition is in a unit dosage form. Preferably, the unit dosage
form is inhaled.
[0082] In another aspect of this embodiment, the pharmaceutical
composition may be co-administered as described above. Preferably,
the BK channel modulator is co-administered with a .beta.-agonist.
It is noted that by using the methods of the present invention,
lower levels of the co-administered composition, e.g.,
.beta.-agonists, may be used; thus reducing the possible side
effects associated with the use of such composition.
[0083] A further embodiment of the present invention is a method
for modulating inflammation in a lung of a patient. This method
comprises administering to a patient an effective of amount of a BK
channel modulator or a pharmaceutical composition comprising a BK
channel modulator, which amount is sufficient to modulate the
inflammation.
[0084] As used herein in relation to inflammation, "modulating",
"modulation" and like terms mean to increase or, preferably, to
decrease inflammation of the lung of a patient administered a
compound or pharmaceutical composition according to the present
invention relative to a patient who is not administered the
compound or the pharmaceutical composition.
[0085] In one aspect of this embodiment, a BK channel modulator or
a pharmaceutical composition comprising a BK channel modulator is
administered in a unit dosage form by e.g., inhalation. In another
aspect of this embodiment, a BK channel modulator or a
pharmaceutical composition comprising a BK channel modulator may be
co-administered as described above. Preferably, the BK channel
modulator is co-administered with a .beta.-agonist.
[0086] Yet another embodiment of the present invention is
pharmaceutical composition for treating or ameliorating the effects
of a disease characterized by altered smooth muscle contractility.
This pharmaceutical composition comprises a pharmaceutically
acceptable carrier and a BK channel modulator.
[0087] In one aspect of this embodiment, diseases characterized by
altered smooth muscle contractility include e.g., pneumoconiosis
(such as aluminosis, anthracosis, asbestosis, chalicosis, ptilosis,
siderosis, silicosis, tabacosis, berylliosis, and byssinosis),
chronic obstructive pulmonary disease (COPD), asthma, bronchitis,
exacerbation of airway hyperreactivity or cystic fibrosis, cough
(including chronic cough), other pulmonary diseases, including
other reversible airway diseases, urinary incontinence, and
hypertension. Preferably, the disease is asthma, chronic
obstructive pulmonary disease, urinary incontinence, or
hypertension. More preferably, the disease is asthma.
[0088] In another aspect of this embodiment, the pharmaceutical
composition is in a unit dosage form. Preferably, the unit dosage
form is inhaled.
[0089] In yet another aspect of this embodiment, the pharmaceutical
composition is co-administered as described above. Preferably, the
BK channel modulator is co-administered with a .beta.-agonist.
[0090] An additional embodiment of the present invention is a
pharmaceutical composition for treating or ameliorating the effects
of asthma. This pharmaceutical composition comprises a
pharmaceutically acceptable carrier and a BK channel modulator.
[0091] A compound or pharmaceutical composition of the present
invention may be administered in any desired and effective manner.
Preferably, the compound or pharmaceutical composition of the
present invention is administered to a patient in need thereof
through a mucosal lining, by, e.g., a nasal or pulmonary spray.
[0092] Thus, compounds and pharmaceutical compositions according to
the present invention may be administered in an aqueous solution as
a nasal or pulmonary spray and may be dispensed in spray form by a
variety of methods known to those skilled in the art. Exemplary
systems for dispensing liquids as a nasal spray are disclosed in
U.S. Pat. No. 4,511,069. The formulations may be presented in
multi-dose containers, for example in the sealed dispensing system
disclosed in U.S. Pat. No. 4,511,069. Additional aerosol delivery
forms may include, e.g., compressed air-, jet-, ultrasonic-, and
piezoelectric nebulizers, which deliver the compound or
pharmaceutical composition according to the present invention
dissolved or suspended in a pharmaceutical solvent, e.g., water,
ethanol, or a mixture thereof.
[0093] For example, a nebulizer may be selected on the basis of
allowing the formation of an aerosol of a BK channel modulator
disclosed herein. The delivered amount of a BK channel modulator
provides a therapeutic effect for the diseases disclosed herein.
The nebulizer may deliver an aerosol comprising a mass median
aerodynamic diameter from about 2 microns to about 5 microns with a
geometric standard deviation less than or equal to about 2.5
microns, a mass median aerodynamic diameter from about 2.5 microns
to about 4.5 microns with a geometric standard deviation less than
or equal to about 1.8 microns, and a mass median aerodynamic
diameter from about 2.8 microns to about 4.3 microns with a
geometric standard deviation less than or equal to about 2 microns.
In other instances, the aerosol can be produced using a vibrating
mesh nebulizer. An example of a vibrating mesh nebulizer includes
the PARI E-FLOW.TM. nebulizer or a nebulizer using PARI eFlow
technology. More examples of nebulizers are provided in U.S. Pat.
Nos. 4,268,460; 4,253,468; 4,046,146; 3,826,255; 4,649,911;
4,510,929; 4,624,251; 5,164,740; 5,586,550; 5,758,637; 6,644,304;
6,338,443; 5,906,202; 5,934,272; 5,960,792; 5,971,951; 6,070,575;
6,192,876; 6,230,706; 6,349,719; 6,367,470; 6,543,442; 6,584,971;
6,601,581; 4,263,907; 5,709,202; 5,823,179; 6,192,876; 6,644,304;
5,549,102; 6,083,922; 6,161,536; 6,264,922; 6,557,549; and
6,612,303; all of which are hereby incorporated by reference in
their entireties. More commercial examples of nebulizers that can
be used with the BK channel modulators described herein include
Respirgard II.TM., Aeroneb.TM., Aeroneb.TM. Pro, and Aeroneb.TM. Go
produced by Aerogen; AERx.TM. and AERx Essence.TM. produced by
Aradigm; Porta-Neb.TM., Freeway Freedom.TM., Sidestream, Ventstream
and I-neb produced by Respironics, Inc. (Murrysville, Pa.); and
PARI LC-Plus.TM., PARI LC-Start, produced by PARI Respiratory
Equipment Inc. (Midlothian, Va.). By further non-limiting example,
U.S. Pat. No. 6,196,219, is hereby incorporated by reference in its
entirety.
[0094] A suitable, non-limiting example of a dosage of a BK channel
modulator according to the present invention administered via a
nebulizer to an adult human may be from about 0.1 mg/m.sup.2/day to
100 mg/m.sup.2/day, such as from about 0.5 mg/m.sup.2/day to about
80 mg/m.sup.2/day, including from about 1 mg/m.sup.2/day to about
50 mg/m.sup.2/day, about 1 mg/m.sup.2/day to about 20
mg/m.sup.2/day, about 1 mg/m.sup.2/day to about 10 mg/m.sup.2/day,
about 1 mg/m.sup.2/day to about 7 mg/m.sup.2/day, or about 3
mg/m.sup.2/day to about 7 mg/m.sup.2/day. Other representative
dosages of a BK channel modulator include about 0.1 mg/m.sup.2/day,
0.2 mg/m.sup.2/day, 0.3 mg/m.sup.2/day, 0.4 mg/m.sup.2/day 0.5
mg/m.sup.2/day, 0.6 mg/m.sup.2/day, 0.7 mg/m.sup.2/day, 0.8
mg/m.sup.2/day, 0.9 mg/m.sup.2/day, 1 mg/m.sup.2/day, 2
mg/m.sup.2/day, 3 mg/m.sup.2/day, 4 mg/m.sup.2/day, 5
mg/m.sup.2/day, 6 mg/m.sup.2/day, 7 mg/m.sup.2/day, 8
mg/m.sup.2/day, 9 mg/m.sup.2/day, 10 mg/m.sup.2/day, 11
mg/m.sup.2/day, 12 mg/m.sup.2/day, 13 mg/m.sup.2/day, 14
mg/m.sup.2/day, 15 mg/m.sup.2/day, 16 mg/m.sup.2/day, 17
mg/m.sup.2/day, 18 mg/m.sup.2/day, 19 mg/m.sup.2/day, 20
mg/m.sup.2/day, 25 mg/m.sup.2/day, 30 mg/m.sup.2/day, 35
mg/m.sup.2/day, 40 mg/m.sup.2/day, 45 mg/m.sup.2/day, 50
mg/m.sup.2/day, 55 mg/m.sup.2/day, 60 mg/m.sup.2/day, 65
mg/m.sup.2/day, 70 mg/m.sup.2/day, 75 mg/m.sup.2/day 80
mg/m.sup.2/day, 85 mg/m.sup.2/day, 90 mg/m.sup.2/day, 95
mg/m.sup.2/day, or 100 mg/m.sup.2/day. Dosages may be reduced in a
child. The effective dose of a BK channel modulator maybe
administered as two, three, four, five, six or more sub-doses,
administered separately at appropriate intervals throughout the
day.
[0095] Nasal and pulmonary spray solutions of the present invention
typically comprise the compound or pharmaceutical composition to be
delivered, optionally formulated with a surface-active agent, such
as a nonionic surfactant (e.g., polysorbate-80), and one or more
buffers. In some embodiments of the present invention, the nasal
spray solution further comprises a propellant. The pH of the nasal
spray solution is optionally between about pH 3.0 and 6.0,
preferably 5.0.+/-0.3. Suitable buffers for use within these
compositions are as described herein or as otherwise known in the
art. Other components may be added to enhance or maintain chemical
stability, including preservatives, surfactants, dispersants, or
gases. Suitable preservatives include, but are not limited to,
phenol, methyl paraben, paraben, m-cresol, thiomersal,
chlorobutanol, benzylalkonimum chloride, and the like. Suitable
surfactants include, but are not limited to, oleic acid, sorbitan
trioleate, polysorbates, lecithin, phosphotidyl cholines, and
various long chain diglycerides and phospholipids. Suitable
dispersants include, but are not limited to,
ethylenediaminetetraacetic acid, and the like. Suitable gases
include, but are not limited to, nitrogen, helium,
chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon
dioxide, air, and the like.
[0096] Within alternate embodiments, mucosal formulations of the
present invention may be administered as dry powder formulations
comprising the compound or pharmaceutical composition according to
the present invention in a dry, usually lyophilized, form of an
appropriate particle size, or within an appropriate particle size
range, for intranasal delivery. Minimum particle size appropriate
for deposition within the nasal or pulmonary passages is often
about 0.5 .mu.m mass median equivalent aerodynamic diameter
(MMEAD), commonly about 1 .mu.m MMEAD, and more typically about 2
.mu.m MMEAD. Maximum particle size appropriate for deposition
within the nasal passages is often about 10 .mu.m MMEAD, commonly
about 8 .mu.m MMEAD, and more typically about 4 .mu.m MMEAD.
Intranasally respirable powders within these size ranges can be
produced by a variety of conventional techniques, such as jet
milling, spray drying, solvent precipitation, supercritical fluid
condensation, and the like. These dry powders of appropriate MMEAD
can be administered to a patient via a conventional dry powder
inhaler (DPI), which rely on the patient's breath, upon pulmonary
or nasal inhalation, to disperse the power into an aerosolized
amount. Alternatively, the dry powder may be administered via
air-assisted devices that use an external power source to disperse
the powder into an aerosolized amount, e.g., a piston pump.
[0097] Dry powder devices typically require a powder mass in the
range from about 1 mg to 20 mg to produce a single aerosolized dose
("puff"). If the required or desired dose of the compound or
pharmaceutical composition according to the present invention is
lower than this amount, the powdered active agent will typically be
combined with a pharmaceutical dry bulking powder to provide the
required total powder mass. Preferred dry bulking powders include
sucrose, lactose, dextrose, mannitol, glycine, trehalose, human
serum albumin (HSA), and starch. Other suitable dry bulking powders
include cellobiose, dextrans, maltotriose, pectin, sodium citrate,
sodium ascorbate, and the like.
[0098] To formulate compositions for mucosal delivery within the
present invention, the compound or pharmaceutical composition
according to the present invention can be combined with various
pharmaceutically acceptable additives, as well as a base or carrier
for dispersion of the active agent(s). Desired additives include,
but are not limited to, pH control agents, such as arginine, sodium
hydroxide, glycine, hydrochloric acid, citric acid, etc. In
addition, local anesthetics (e.g., benzyl alcohol), isotonizing
agents (e.g., sodium chloride, mannitol, sorbitol), adsorption
inhibitors (e.g., Tween 80), solubility enhancing agents (e.g.,
cyclodextrins and derivatives thereof), stabilizers (e.g., serum
albumin), and reducing agents (e.g., glutathione) can be included.
When the composition for mucosal delivery is a liquid, the tonicity
of the formulation, as measured with reference to the tonicity of
0.9% (w/v) physiological saline solution taken as unity, is
typically adjusted to a value at which no substantial, irreversible
tissue damage will be induced in the nasal mucosa at the site of
administration. Generally, the tonicity of the solution is adjusted
to a value of about 1/3 to 3, more typically 1/2 to 2, and most
often 3/4 to 1.7.
[0099] The compounds or compositions of the present invention may
be dispersed in a base or vehicle, which may comprise a hydrophilic
compound having a capacity to disperse the compounds or
compositions of the present invention and any desired additives.
The base may be selected from a wide range of suitable carriers,
including but not limited to, copolymers of polycarboxylic acids or
salts thereof, carboxylic anhydrides (e.g. maleic anhydride) with
other monomers (e.g. methyl (meth)acrylate, acrylic acid, etc.),
hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl
alcohol, polyvinylpyrrolidone, cellulose derivatives such as
hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural
polymers such as chitosan, collagen, sodium alginate, gelatin,
hyaluronic acid, and nontoxic metal salts thereof. Often, a
biodegradable polymer is selected as a base or carrier, for
example, polylactic acid, poly(lactic acid-glycolic acid)
copolymer, polyhydroxybutyric acid, poly(hydroxybutyric
acid-glycolic acid) copolymer and mixtures thereof. Alternatively
or additionally, synthetic fatty acid esters such as polyglycerin
fatty acid esters, sucrose fatty acid esters, etc. can be employed
as carriers. Hydrophilic polymers and other carriers can be used
alone or in combination, and enhanced structural integrity can be
imparted to the carrier by partial crystallization, ionic bonding,
crosslinking and the like. The carrier can be provided in a variety
of forms, including, fluid or viscous solutions, gels, pastes,
powders, microspheres and films for direct application to the nasal
mucosa. The use of a selected carrier in this context may result in
promotion of absorption of the compound or composition according to
the present invention.
[0100] The compounds or compositions of the present invention can
be combined with the base or carrier according to a variety of
methods, and release of the compounds or compositions of the
present invention may be by diffusion, disintegration of the
carrier, or associated formulation of water channels. In some
circumstances, the active agent is dispersed in microcapsules
(microspheres) or nanocapsules (nanospheres) prepared from a
suitable polymer, e.g., isobutyl 2-cyanoacrylate and dispersed in a
biocompatible dispersing medium applied to the nasal mucosa, which
yields sustained delivery and biological activity over a protracted
time.
[0101] To further enhance mucosal delivery of compounds or
compositions of the present invention, formulations comprising such
agents may also contain a hydrophilic low molecular weight compound
as a base or excipient. Such hydrophilic low molecular weight
compounds provide a passage medium through which a water-soluble
active agent, such as a physiologically active peptide or protein,
may diffuse through the base to the body surface where the active
agent is absorbed. The hydrophilic low molecular weight compound
optionally absorbs moisture from the mucosa or the administration
atmosphere and dissolves the water-soluble active peptide. The
molecular weight of the hydrophilic low molecular weight compound
is generally not more than 10,000 and preferably not more than
3,000. Exemplary hydrophilic low molecular weight compounds include
polyol compounds, such as oligo-, di- and monosaccarides such as
sucrose, mannitol, sorbitol, lactose, L-arabinose, D-erythrose,
D-ribose, D-xylose, D-mannose, trehalose, D-galactose, lactulose,
cellobiose, gentibiose, glycerin and polyethylene glycol. Other
examples of hydrophilic low molecular weight compounds useful as
carriers within the invention include N-methylpyrrolidone, and
alcohols (e.g. oligovinyl alcohol, ethanol, ethylene glycol,
propylene glycol, etc.) These hydrophilic low molecular weight
compounds can be used alone or in combination with one another or
with other active or inactive components of the intranasal
formulation.
[0102] In sum, mucosal administration according to the invention
allows effective self-administration of treatment by patients,
provided that sufficient safeguards are in place to control and
monitor dosing and side effects. Mucosal administration also
overcomes certain drawbacks of other administration forms, such as
injections, that are painful and expose the patient to possible
infections and may present drug bioavailability problems. For nasal
and pulmonary delivery, systems for controlled aerosol dispensing
of therapeutic liquids as a spray are well known. For example,
metered doses of a compound or composition of the present invention
are delivered by means of a specially constructed mechanical pump
valve, U.S. Pat. No. 4,511,069.
[0103] In the present invention, other methods of delivery may also
be used. Such methods include, for example, administration by oral
ingestion, or as an ointment or drop for local administration to
the eyes, or for parenteral or other administration in any
appropriate manner such as intraperitoneal, subcutaneous, topical,
intradermal, rectal, vaginal, sublingual, intramuscular,
intravenous, intraarterial, intrathecal, or intralymphatic.
Further, a pharmaceutical composition of the present invention may
be administered in conjunction with other treatments. A
pharmaceutical composition of the present invention may be
encapsulated or otherwise protected against gastric or other
secretions, if desired.
[0104] The pharmaceutically acceptable compositions of the
invention comprise one or more active ingredients in admixture with
one or more pharmaceutically-acceptable carriers and, optionally,
one or more other compounds, drugs, ingredients and/or materials.
Regardless of the route of administration selected, the
agents/compounds of the present invention are formulated into
pharmaceutically-acceptable dosage forms by conventional methods
known to those of skill in the art. See, e.g., Remington, The
Science and Practice of Pharmacy (21.sup.st Edition, Lippincott
Williams and Wilkins, Philadelphia, Pa.).
[0105] Pharmaceutically acceptable carriers are well known in the
art (see, e.g., Remington, The Science and Practice of Pharmacy
(21.sup.st Edition, Lippincott Williams and Wilkins, Philadelphia,
Pa.) and The National Formulary (American Pharmaceutical
Association, Washington, D.C.)) and include sugars (e.g., lactose,
sucrose, mannitol, and sorbitol), starches, cellulose preparations,
calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate
and calcium hydrogen phosphate), sodium citrate, water, aqueous
solutions (e.g., saline, sodium chloride injection, Ringer's
injection, dextrose injection, dextrose and sodium chloride
injection, lactated Ringer's injection), alcohols (e.g., ethyl
alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g.,
glycerol, propylene glycol, and polyethylene glycol), organic
esters (e.g., ethyl oleate and tryglycerides), biodegradable
polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and
poly(anhydrides)), elastomeric matrices, liposomes, microspheres,
oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and
groundnut), cocoa butter, waxes (e.g., suppository waxes),
paraffins, silicones, talc, silicylate, etc. Each pharmaceutically
acceptable carrier used in a pharmaceutical composition of the
invention must be "acceptable" in the sense of being compatible
with the other ingredients of the formulation and not injurious to
the subject. Carriers suitable for a selected dosage form and
intended route of administration are well known in the art, and
acceptable carriers for a chosen dosage form and method of
administration can be determined using ordinary skill in the
art.
[0106] The pharmaceutical compositions of the invention may,
optionally, contain additional ingredients and/or materials
commonly used in such pharmaceutical compositions. These
ingredients and materials are well known in the art and include (1)
fillers or extenders, such as starches, lactose, sucrose, glucose,
mannitol, and silicic acid; (2) binders, such as
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants,
such as glycerol; (4) disintegrating agents, such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, sodium starch glycolate, cross-linked sodium
carboxymethyl cellulose and sodium carbonate; (5) solution
retarding agents, such as paraffin; (6) absorption accelerators,
such as quaternary ammonium compounds; (7) wetting agents, such as
cetyl alcohol and glycerol monostearate; (8) absorbents, such as
kaolin and bentonite clay; (9) lubricants, such as talc, calcium
stearate, magnesium stearate, solid polyethylene glycols, and
sodium lauryl sulfate; (10) suspending agents, such as ethoxylated
isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth; (11) buffering agents; (12) excipients,
such as lactose, milk sugars, polyethylene glycols, animal and
vegetable fats, oils, waxes, paraffins, cocoa butter, starches,
tragacanth, cellulose derivatives, polyethylene glycol, silicones,
bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum
hydroxide, calcium silicates, and polyamide powder; (13) inert
diluents, such as water or other solvents; (14) preservatives; (15)
surface-active agents; (16) dispersing agents; (17) control-release
or absorption-delaying agents, such as hydroxypropylmethyl
cellulose, other polymer matrices, biodegradable polymers,
liposomes, microspheres, aluminum monosterate, gelatin, and waxes;
(18) opacifying agents; (19) adjuvants; (20) wetting agents; (21)
emulsifying and suspending agents; (22), solubilizing agents and
emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan; (23) propellants as disclosed above, such
as hydrofluoroalkane, particularly 1,1,1,2-tetrafluoroethane,
heptafluoralkane (HFA) such as 1,1,1,2,3,3,3-heptafluoro-n-propane
or mixtures thereof, as well as other chlorofluorohydrocarbons and
other volatile unsubstituted hydrocarbons, such as butane and
propane; (24) antioxidants; (25) agents which render the
formulation isotonic with the blood of the intended recipient, such
as sugars and sodium chloride; (26) thickening agents; (27) coating
materials, such as lecithin; and (28) sweetening, flavoring,
coloring, perfuming and preservative agents. Each such ingredient
or material must be "acceptable" in the sense of being compatible
with the other ingredients of the formulation and not injurious to
the subject. Ingredients and materials suitable for a selected
dosage form and intended route of administration are well known in
the art, and acceptable ingredients and materials for a chosen
dosage form and method of administration may be determined using
ordinary skill in the art.
[0107] Pharmaceutical compositions suitable for oral administration
may be in the form of capsules, cachets, pills, tablets, powders,
granules, a solution or a suspension in an aqueous or non-aqueous
liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir
or syrup, a pastille, a bolus, an electuary or a paste. These
formulations may be prepared by methods known in the art, e.g., by
means of conventional pan-coating, mixing, granulation or
lyophilization processes.
[0108] Solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules and the like) may be
prepared, e.g., by mixing the active ingredient(s) with one or more
pharmaceutically-acceptable carriers and, optionally, one or more
fillers, extenders, binders, humectants, disintegrating agents,
solution retarding agents, absorption accelerators, wetting agents,
absorbents, lubricants, and/or coloring agents. Solid compositions
of a similar type may be employed as fillers in soft and
hard-filled gelatin capsules using a suitable excipient. A tablet
may be made by compression or molding, optionally with one or more
accessory ingredients. Compressed tablets may be prepared using a
suitable binder, lubricant, inert diluent, preservative,
disintegrant, surface-active or dispersing agent. Molded tablets
may be made by molding in a suitable machine. The tablets, and
other solid dosage forms, such as dragees, capsules, pills and
granules, may optionally be scored or prepared with coatings and
shells, such as enteric coatings and other coatings well known in
the pharmaceutical-formulating art. They may also be formulated so
as to provide slow or controlled release of the active ingredient
therein. They may be sterilized by, for example, filtration through
a bacteria-retaining filter. These compositions may also optionally
contain opacifying agents and may be of a composition such that
they release the active ingredient only, or preferentially, in a
certain portion of the gastrointestinal tract, optionally, in a
delayed manner. The active ingredient can also be in
microencapsulated form.
[0109] Liquid dosage forms for oral administration include
pharmaceutically-acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs. The liquid dosage forms may
contain suitable inert diluents commonly used in the art. Besides
inert diluents, the oral compositions may also include adjuvants,
such as wetting agents, emulsifying and suspending agents,
sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions may contain suspending agents.
[0110] Pharmaceutical compositions for rectal or vaginal
administration may be presented as a suppository, which may be
prepared by mixing one or more active ingredient(s) with one or
more suitable nonirritating carriers which are solid at room
temperature, but liquid at body temperature and, therefore, will
melt in the rectum or vaginal cavity and release the active
compound. Pharmaceutical compositions which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such
pharmaceutically-acceptable carriers as are known in the art to be
appropriate.
[0111] Dosage forms for the topical or transdermal administration
include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches, drops and inhalants as previously disclosed.
The active agent(s)/compound(s) may be mixed under sterile
conditions with a suitable pharmaceutically-acceptable carrier. The
ointments, pastes, creams and gels may contain excipients. Powders
and sprays may contain excipients and propellants as previously
disclosed.
[0112] Pharmaceutical compositions suitable for parenteral
administrations comprise one or more agent(s)/compound(s) in
combination with one or more pharmaceutically-acceptable sterile
isotonic aqueous or non-aqueous solutions, dispersions, suspensions
or emulsions, or sterile powders which may be reconstituted into
sterile injectable solutions or dispersions just prior to use,
which may contain suitable antioxidants, buffers, solutes which
render the formulation isotonic with the blood of the intended
recipient, or suspending or thickening agents. Proper fluidity can
be maintained, for example, by the use of coating materials, by the
maintenance of the required particle size in the case of
dispersions, and by the use of surfactants. These compositions may
also contain suitable adjuvants, such as wetting agents,
emulsifying agents and dispersing agents. It may also be desirable
to include isotonic agents. In addition, prolonged absorption of
the injectable pharmaceutical form may be brought about by the
inclusion of agents which delay absorption.
[0113] In some cases, in order to prolong the effect of a drug
(e.g., pharmaceutical formulation), it is desirable to slow its
absorption from subcutaneous or intramuscular injection. This may
be accomplished by the use of a liquid suspension of crystalline or
amorphous material having poor water solubility.
[0114] The rate of absorption of the active agent/drug then depends
upon its rate of dissolution which, in turn, may depend upon
crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered agent/drug may be
accomplished by dissolving or suspending the active agent/drug in
an oil vehicle. Injectable depot forms may be made by forming
microencapsule matrices of the active ingredient in biodegradable
polymers. Depending on the ratio of the active ingredient to
polymer, and the nature of the particular polymer employed, the
rate of active ingredient release can be controlled. Depot
injectable formulations are also prepared by entrapping the drug in
liposomes or microemulsions which are compatible with body tissue.
The injectable materials can be sterilized for example, by
filtration through a bacterial-retaining filter.
[0115] The formulations may be presented in unit-dose or multi-dose
sealed containers, for example, ampules and vials, and may be
stored in a lyophilized condition requiring only the addition of
the sterile liquid carrier, for example water for injection,
immediately prior to use. Extemporaneous injection solutions and
suspensions may be prepared from sterile powders, granules and
tablets of the type described above.
[0116] As described above, BK.sub.Ca channels play a critical role
in modulating neuronal processes and smooth muscle contractile
tone. Accordingly, BK.sub.Ca regulation has significant
implications in the study of diseases in which smooth muscle
contraction may be abnormal. Alteration of the channel's activity
by phosphorylation represents an important regulatory pathway
leading to modulation of cellular excitability. The present
inventors have herein demonstrated that pharmacologic approaches to
activate BK.sub.Ca channels represent an emerging novel strategy to
control membrane excitability.
[0117] Thus, in another aspect, the present invention relates to
several findings, concerning BK.sub.Ca regulation. In particular,
the inventors have discovered that rottlerin dramatically increases
BK.sub.Ca channel activity in a non-Ca.sup.2+ dependent, but
reversible fashion. Moreover, rottlerin's mechanism appears unique:
tail currents are markedly prolonged after exposure to rottlerin,
implying a slowing of deactivation and the G-V curve is reversibly
shifted by more than 100 mV to the left. Similar results were
observed in a rat BK.sub.Ca channel heterologously expressed in
HEK293. Accordingly, the present invention provides compositions
and methods for regulating the BK channel using rottlerin and
derivatives thereof.
[0118] More specifically, the present invention also encompasses
compositions and methods for treating or preventing BK channel
mediated disorders by administering to a subject an effective
amount of a BK channel activator, including but not limited to
rottlerin and derivatives thereof.
[0119] As used herein, a BK channel or BK.sub.Ca channel mediated
disorder refers to disorders related to under or over activation of
the BK channel. For purposes of the present invention, such
disorders include, but are not limited to, hypertension, asthma,
urinary incontinence, gastroenteric hypermotility, coronary spasm,
pulmonary disease, psychoses, convulsion, anxiety, erectile
dysfunction and neurologic dysfunction.
[0120] The term "derivative" as used herein refers to a chemical
compound that is structurally similar to another and may be
theoretically derivable from it, but differs slightly in
composition, but has the same or better activity and safety
profile. For example, an analogue of rottlerin is a compound that
differs slightly from rottlerin (e.g., as in the replacement of one
atom by an atom of a different element or in the presence of a
particular functional group), and may be derivable from
rottlerin.
[0121] In one aspect of the present invention, a compound for use
in modulating BK channel activity is provided having the general
formula:
##STR00001##
wherein X is CH.sub.2, O, N, or S; R.sub.1 and R.sub.3 are
independently selected from H, OH, NH or SH; R.sub.2 is ethanone,
acetyl, alkenyl, aryl or alkyl; R.sub.4 is CO-[(E)CHCH].sub.n-Ph,
CN-[(E)CHCH].sub.n-Ph, or COOZ, wherein Z is alkenyl, aryl, or
alkyl; and R.sub.5 and R.sub.6 are independently selected from H,
OH, NH, SH, alkenyl, aryl, or alkyl. In one embodiment, the
compound is rottlerin or a derivative thereof.
[0122] The present invention also provides a pharmaceutical
composition comprising the above-described compound, or a
derivative thereof, and optionally, a pharmaceutically acceptable
carrier, for use in treating or preventing a BK channel associated
disorder. In a specific embodiment, the compound is rottlerin.
[0123] The term "treating," as used herein in relation to a
disorder (as opposed to the effects of a disorder), includes
treating any one or more of the conditions underlying or
characteristic of a particular disorder. As used herein, the term
"preventing" in relation to a disorder (as opposed to the effects
of a disorder), includes preventing the initiation of a particular
disorder, delaying the initiation the disorder, preventing the
progression or advancement of the disorder, slowing the progression
or advancement of the disorder, delaying the progression or
advancement of the disorder, and reversing the progression of the
disorder from an advanced to a less advanced stage.
[0124] By way of example, in an embodiment of the invention,
hypertension is treated in a subject in need of treatment by
administering to the subject a therapeutically effective amount of
rottlerin or a derivative thereof, which amount is effective to
treat the hypertension. The subject is preferably a mammal (e.g.,
humans, domestic animals, and commercial animals, including cows,
dogs, monkeys, mice, pigs, and rats), and is most preferably a
human.
[0125] In another aspect of the present invention, the
above-described compounds and pharmaceutical compositions can be
used to regulate membrane excitability both in vitro and in vivo.
In one example, the compounds and pharmaceutical compositions of
the present invention can be used to treat or prevent a
hyperexcitability disorder. In an embodiment of the invention, the
hyperexcitability disorder is asthma. In another embodiment of the
invention, the hyperexcitability disorder is hypertension. In other
embodiments of the present invention, the hyperexcitability
disorder includes, but is not necessarily limited to, urinary
incontinence, gastroenteric hypermotility, coronary spasm,
psychoses, convulsion and anxiety. In another embodiment, the
compounds and pharmaceutical compositions of the present invention
are used in treating or preventing erectile dysfunction. In yet
other embodiments, the compounds and pharmaceutical compositions of
the present invention are used in treating or preventing coronary
artery vasospasm. In another embodiment, the compounds and
pharmaceutical compositions of the present invention are used in
treating or preventing neurologic dysfunction. In another
embodiment, the compounds and pharmaceutical compositions of the
present invention are used in post-stroke neuroprotection.
[0126] The present invention also provides methods for treating or
preventing a hyperexcitability disorder in a subject, comprising
administering to the subject a therapeutically effective amount of
the pharmaceutical composition of the invention. In an embodiment
of the invention, the hyperexcitability disorder includes, but is
not necessarily limited to, asthma, urinary incontinence,
gastroenteric hypermotility, hypertension, coronary spasm,
psychoses, convulsion and anxiety.
[0127] The present invention also provides methods for treating or
preventing erectile dysfunction in a subject by administering to
the subject a therapeutically effective amount of the
pharmaceutical compositions of the invention. Additionally, the
present invention also provides methods for treating or preventing
a coronary artery vasospasm in a subject, comprising administering
to the subject a therapeutically effective amount of a
pharmaceutical composition of the present invention.
[0128] The present invention additionally provides methods for
treating or preventing hypertension in a subject, comprising
administering to the subject a therapeutically effective amount of
a pharmaceutical composition of the present invention. As used in
the context of the present invention, hypertension refers to a
condition characterized by an increased systolic and/or diastolic
blood pressure. By way of non-limiting example, hypertension in a
human subject is characterized by a systolic pressure above 140 mm
Hg and/or a diastolic pressure above 90 mm Hg.
[0129] The present invention also provides methods for treating or
preventing a neurologic dysfunction in a subject, comprising
administering to the subject a therapeutically effective amount of
a pharmaceutical composition of the present invention. The present
invention also provides methods for post-stroke neuroprotection in
a subject by administering a therapeutically effective amount of a
pharmaceutical composition of the present invention.
[0130] The present invention further provides kits for use in
treating or preventing hyperexcitability disorders comprising an
effective amount of a pharmaceutical composition of the present
invention, optionally, in association with a pharmaceutically
acceptable carrier. In an embodiment, the hyperexcitability
disorder includes, but is not necessarily limited to, asthma,
urinary incontinence, gastroenteric hypermotility, hypertension,
coronary spasm, psychoses, convulsion and anxiety.
[0131] The present invention also provides kits for use in treating
or preventing erectile dysfunction, coronary artery vasospasm,
hypertension or neurologic dysfunction in a subject, comprising
administering a therapeutically effective amount of a
pharmaceutical composition of the present invention.
[0132] Finally, the present invention also provides kits for use in
post-stroke neuroprotection in a subject, comprising a
therapeutically effective amount of a pharmaceutical composition of
the present invention.
[0133] As noted above, rottlerin
(5,7-dihydroxy-2,2-dimethyl-6-(2,4,6-trihydroxy-3-methyl-5-acetylbenzyl)--
8-cinnamoyl-I,2-chromene), and derivatives thereof, have been
frequently, but incorrectly, characterized in the literature as
PKC.delta. inhibitors. The present invention establishes for the
first time that rottlerin and its derivatives can be used to
activate the BK.sub.Ca channel. This new therapy will provide
unique strategies to treat and prevent a variety of disorders
mediated by BK.sub.Ca channel activity.
[0134] Methods of preparing rottlerin and its derivatives are well
known in the art. Rottlerin, for example, is commercially available
from A.G. Scientific, Inc., 6450 Lusk Blvd: Suite E 102, San Diego,
Calif. 92121. Rottlerin and derivatives thereof may be synthesized
in accordance with known organic chemistry procedures that are
readily understood by those of skill in the art. The term
"synthesize" as used in the present invention refers to formation
of a particular chemical compound from its constituent parts using
synthesis processes known in the art. Such synthesis processes
include, for example, the use of light, heat, chemical, enzymatic
or other means to form particular chemical composition.
[0135] In a method of the present invention, a composition
comprising rottlerin or a derivative thereof may be administered to
a subject in combination with another BK.sub.Ca channel activator,
such that a synergistic therapeutic effect is produced. A
"synergistic therapeutic effect" refers to a greater-than-additive
therapeutic effect which is produced by a combination of two
therapeutic agents, and which exceeds that which would otherwise
result from individual administration of either therapeutic agent
alone. For instance, administration of rottlerin in combination
with a derivative thereof unexpectedly results in a synergistic
therapeutic effect by providing greater efficacy than would result
from use of either of therapeutic agents alone. Rottlerin enhances
the effect of the rottlerin derivative. Therefore, lower doses of
one or both of the therapeutic agents may be used in treating for
example, hypertension, resulting in increased therapeutic efficacy
and decreased side-effects.
[0136] The invention also provides compositions and methods for
treating or preventing neuronal damage in a post-stroke subject
comprising administering to the subject a therapeutically effective
amount of rottlerin or derivatives thereof.
[0137] The present invention further provides kits for use in
treating or preventing hyperexcitability disorders in a subject
comprising a therapeutically effective amount of a pharmaceutical
composition of the present invention, optionally, in combination
with a pharmaceutically acceptable carrier. In an embodiment, the
hyperexcitability disorder includes, but is not necessarily limited
to, asthma, urinary incontinence, gastroenteric hypermotility,
hypertension, coronary spasm, psychoses, convulsion and
anxiety.
[0138] The present invention also provides kits for use in treating
or preventing erectile dysfunction, coronary artery vasospasm,
hypertension or neurologic dysfunction in a subject, comprising
administering a therapeutically effective amount of a
pharmaceutical composition of the present invention. The present
invention further provides kits for use in treating or preventing
hyperexcitability disorders in a subject comprising a
therapeutically effective amount of a pharmaceutical composition of
the present invention, optionally, in combination with a
pharmaceutically acceptable carrier. In an embodiment, the
hyperexcitability disorder includes, but is not necessarily limited
to, asthma, urinary incontinence, gastroenteric hypermotility,
hypertension, coronary spasm, psychoses, convulsion, and
anxiety.
[0139] The present invention also provides pharmaceutical
compositions for use in treating or preventing erectile
dysfunction, coronary artery vasospasm, hypertension or neurologic
dysfunction in a subject, comprising administering a
therapeutically effective amount of a pharmaceutical composition of
the present invention.
[0140] Finally, the present invention also provides kits for use in
post-stroke neuroprotection in a subject, comprising a
therapeutically effective amount of a pharmaceutical composition of
the present invention.
[0141] The following examples are provided to further illustrate
the compositions and methods of the present invention. These
examples are illustrative only and are not intended to limit the
scope of the invention in any way.
EXAMPLES
Example 1
Rottlerin Activates BK.sub.Ca Channel
[0142] BK.sub.Ca regulation has significant implications in the
study of diseases in which smooth muscle contraction may be
abnormal. BK.sub.Ca can be potently regulated by PKC activating
vasoconstrictors. In order to elucidate the functional effects of
PKC phosphorylation, the inventors evaluated putative PKC
inhibitory compounds under non-phosphorylated conditions. Thus, it
is expected that these PKC inhibitors should have no effect in this
study.
[0143] Surprisingly, one compound, rottlerin (<100 nM)
dramatically increased channel activity (FIG. 4) in a non-Ca.sup.2+
dependent, but reversible fashion. No other PKC inhibitor had any
effect on BK.sub.Ca channel activity under basal conditions (not
shown). Moreover, rottlerin's mechanism appears unique; tail
currents are markedly prolonged after exposure to rottlerin,
implying a slowing of deactivation, and the G-V curve is reversibly
shifted by more than 100 mV to the left (FIG. 4B). Similar results
were observed in a rat BK.sub.Ca channel heterologously expressed
in HEK293. Intracellular dialysis of rottlerin (the inventors
tested up to 20 .mu.M, which represents .about.200 fold more than
the maximum extracellular concentration tested) had only a
relatively small effect on the channel activity, suggesting that
access to the activating site requires extracellular exposure (FIG.
5).
[0144] Although rottlerin has been proposed to have other effects
at significantly high concentrations (.about.10 .mu.M), the
BK.sub.Ca activation is not due to modulation by PKC (or other
cellular components), because it can be demonstrated using a
cell-free configuration (FIG. 6).
[0145] Rottlerin was compared to one of the originally described
BK.sub.Ca channel activators, NS-1619 (Olesen, et ah, Selective
activation of Ca.sup.2+-dependent K.sup.+ channels by novel
benzimidazolone. Eur. J. Pharmacol, 1994. 251(1):53-9). NS-1619 (10
.mu.M) activated peak K.sup.+ current in HEK293 cells stably
transfected with mSlo (FIG. 7A); addition of rottlerin (0.5 .mu.M)
after NS-1619 administration incrementally increased current in 0
Ca.sup.2+. Co-expression of the .beta.1 subunit does not modify the
effects of rottlerin. In HEK293 cells expressing both mSlo and
.beta.1 subunit, rottlerin activated the channel (FIG. 7B).
[0146] Given rottlerin's potent BK.sub.Ca activating effects, in
the absence of Ca.sup.2+ and the presence of the .beta.1 subunit,
the inventors hypothesized that rottlerin may be effective in
mediating the relaxation of vascular smooth muscle. Human vascular
smooth muscle cells (VSMC) grown in vitro express BK.sub.Ca
channels. Single channel recordings (outside-out configuration)
demonstrate that rottlerin activated BK.sub.Ca channels (FIG. 7C)
by increasing Po and open dwell time.
[0147] Next, the inventors determined whether rottlerin could
mediate vascular relaxation, as demonstrated for several other
BK.sub.Ca channel activators (Nardi, et al., Natural modulators of
large-conductance calcium-activated potassium channels. Phnta.
Med., 2003. 69(10):885-92). Rottlerin (4 .mu.M) reduced
phenylephrine mediated contraction by more than 50% (FIG. 7D),
although 1 .mu.M had no significant effect. The rottlerin mediated
effect was inhibited by TEA, suggesting a prominent K.sup.+ channel
contribution to the blunting of contractile tone. The difference in
concentration between the BK.sub.Ca channel effects in
electrophysiologic experiments compared to vascular rings may be
explained by the hydrophobic properties of the compound and the
experimental conditions. Interestingly, NS-1619's efficacy in
mediating vascular relaxation was diminished in a hypertensive rat
model (Callera, et al., Ca.sup.2+-activated K.sup.+ channels
underlying the impaired acetylcholine-induced vasodilation in 2K-1C
hypertensive rats. J. Pharmacol. Exp. Ther., 2004. 309(3):
1036-42), compared to controls, perhaps consistent with the
down-regulation of the .beta.1, but not .alpha. subunit observed in
hypertensive animal models (Amberg, et al., Downregulation of the
BK channel .beta.1 subunit in genetic hypertension. Circ. Res.,
2003. 93(10):965-71; Amberg, et al., Modulation of the molecular
composition of large conductance, Ca.sup.2+ activated K.sup.+
channels in vascular smooth muscle during hypertension. J. Clin.
Invest., 2003. 112(5):717-24). Based upon rottlerin's efficacy in
in vitro electrophysiologic experiments (FIG. 4-7), in low
[Ca.sup.2+].sub.i and in the absence of a .beta.1 subunit, the
inventors hypothesize that rottlerin-like compounds may be more
effective.
Example 2
[0148] Rottlerin-Induced Activation of BK Channels does not Involve
Phosphorylation or Cytosolic Components
[0149] Murine tracheal smooth muscle cells were isolated using the
following protocol. Trachea were removed, cut longitudinally, the
epithelium removed (brushing with sterile cotton bud) and the
cartilage removed by cutting. The isolated trachea were dissected
in culture medium and cut into several pieces (1-2 mm.sup.2). After
addition of 0.5 mg/ml papain (Roche, Nutley, N.J.) and 1 mg/ml
dithiothreitol, the cells were dissociated at 37.degree. C. for 20
minutes, gently shaking, followed by the addition of 0.1 mg/ml
liberase enzyme 2 (Roche, Nutley, N.J.) for 30 minutes at
37.degree. C. (5% CO.sub.2). The suspension was then pipetted
gently several times to disburse cells, the cell suspension
strained via a nylon cell strainer, and the suspension was gently
triturated to disburse single cells. The cell suspension was then
centrifuged at 700.times.g for 5 minutes and the pellet resuspended
in 500 .mu.l Krebs solution. The cells were plated onto
laminin-coated tissue culture dishes. BK currents were recorded
using whole-cell and outside-out macropatches.
[0150] As shown in FIG. 11A, rottlerin (1 mM) significantly
activated BK currents in tracheal smooth muscle. When measuring the
membrane potential, a significant hyperpolarization of the membrane
was observed after rottlerin application, which was completely
reversed after administration of paxilline, a BK channel antagonist
(FIGS. 11B and 11D).
[0151] Rottlerin's ability to induce hyperpolarization of the
membrane potential of tracheal smooth muscle cells was confirmed by
another set of experiments. The membrane potential was recorded
using perforated patch clamp technique. As shown in FIG. 15,
rottlerin induced significant hyperpolarization of the membrane
potential of tracheal smooth muscle.
[0152] In outside-out patches pulled from HEK cells stably
expressing BK channels and in cultured human VSMC, bath application
of 0.5 .mu.M rottlerin resulted in a significant increase in
channel open probability (FIG. 12), which was reversed with
wash-out (not shown). The reversible activation of BK channels by
rottlerin in a cell-free configuration, in the absence of ATP,
implies that rottlerin-induced activation of BK channels does not
involve phosphorylation or cytosolic components.
Example 3
[0153] Isoproterenol-Induced Relaxation was Dependent Upon BK
Channel-Induced Hyperpolarization
[0154] It is well-known that .beta.-adrenergic agonists promote
relaxation of airway smooth muscle. Inhibition of BK channels has
been shown to reduce .beta.-adrenergic agonist-induced
relaxation.
[0155] To dissect which (or both) .beta.-AR pathways are
responsible for the effects, tracheal rings were pre-incubated with
either .beta.1-AR antagonist (CGP 20712A; 100 nM) or a .beta.2-AR
antagonist (ICI 118551; 100 nM) or vehicle (DMSO). FIG. 8A shows
that pre-incubation of rings with .beta.2-AR antagonist resulted in
a rightward shift in the dose-response curve indicating that
.beta.2AR pathway is primarily responsible for the relaxation. To
confirm that the isoproterenol-induced relaxation was dependent
upon BK channel-induced hyperpolarization, IbTX, a specific
inhibitor of the BK channel, was used. FIG. 8B shows that in the
presence of IbTX, tracheal rings of WT showed a significant
decrease in relaxation in response to isoproterenol, confirming
prior published results.
[0156] The effect of ISO on BK currents in acutely isolated
tracheal ASM was also determined. ASM cells were studied using
perforated whole cell voltage clamp. After obtaining access,
outward K.sup.+ current was monitored using a 0.5 second ramp, from
a holding potential of -20 mV to +200 mV. After recording a stable
baseline, the cells were exposed to 0.5 .mu.M ISO, which increased
outward K.sup.+ current by more than 50%, which was substantially
blocked by IbTX (FIGS. 23A and 23B). Under the conditions used, the
majority of the outward K.sup.+ current is conducted by the BK
channel, as shown by IbTX-blockade (FIGS. 23A and 23B).
[0157] To test the hypothesis that rottlerin-induced
hyperpolarization causes relaxation of ASM, rottlerin was added on
tracheal rings mounted on a myograph, and ISO-induced relaxation
was measured. Rottlerin enhanced the ISO-induced relaxation of
tracheal rings when compared to PBS, shifting the ISO-relaxation
curve upward and to the left (FIG. 11C). However, tracheal rings
pre-incubated with iberiotoxin (IbTx), a BK channel inhibitor,
showed minimal relaxation to ISO, even in the presence of rottlerin
further indicating rottlerin's effect via BK channels.
Example 4
Administration of Rottlerin in Asthma Model
[0158] In this example, the acute asthma model in ovalbumin (OVA)
treated mice was used (Jia, Y., R. Foronjy, and J. D'Armiento,
Altered airway inflammatory response to cigarette smoke in
ovalbumin-sensitized mice. Am J. Resp and Critical Care Medicine
(2006) p. Suppl:A339.). Rottlerin was administered to asthmatic
mice to test whether it attenuated the development of asthma. Mice
received an intraperitoneal injection (i.p) of 100 .mu.g ovalbumin
adsorbed to 2 mg aluminum (200 .mu.l final volume) on day 0 and
again on day 7. Control mice were injected with endotoxin free PBS.
On alternative days 14-22, mice received a 20 minute aerosol
challenge of either endotoxin-free PBS (controls) or 2% (w/v) OVA
in endotoxin-PBS, using a lumiscope 6610 ultrasonic nebulizer
(Lumiscope, East Rutherford, N.J.). For rottlerin (Sigma, St.
Louis, Mo.) treatment mice received one i.p injection of 5 mg per
kg body weight on day 13, one injection an hour before each aerosol
challenge and one before methacholine challenge.
[0159] Two days after the last aerosol challenge, AHR was measured
by invasive restrained whole body plethysmography (rWBP) (BUXCO
Electronics Inc., Troy, N.Y.) in response to inhaled methacholine
(Sigma, St. Louis, Mo.). For dynamic lung resistance, measurements
were performed using the PLY3011 chamber (BUXCO). Mice were
anesthetized with a cocktail of 25 mg per kg body weight ketamine
hydrochloride (Bioniche Pharma, Lake Forest, Ill.) and 2.5 mg per
kg body weight xylazine (Llyod Laboratories, Inc., Barangay Tikay,
Malolos Bulacan, Philippines), tracheotomized, and immediately
intubated with an 18-gauge catheter, followed by mechanical
ventilation (Columbus Instruments International, Columbus, Ohio).
Respiratory frequency was set at 150 breaths/minute with a tidal
volume of 0.2 ml. Increasing concentrations of methacholine (0-50
mg/ml) were administered at the rate of 20 puffs per 10 seconds,
with each puff of aerosol delivery lasting 10 ms, via a nebulizer
aerosol system with a 4-6 .mu.m aerosol particle size generated by
a nebulizer head (Aeroneb.RTM., Aerogen Ltd., Dangan, Galway,
Ireland). The nebulizer is attached to a small aerosol block, which
is placed in the inspiratory flow line. In this way the aerosol is
injected directly into the airway. Baseline resistance was restored
before administration of the subsequent doses of methacholine. The
flow and pressure signals were measured and processed together to
determine resistance and compliance using a software analyzer
provided in BioSystem XA software (BUXCO Electronics Inc., Troy,
N.Y.).
[0160] After measurement of airway responsiveness in vivo, mice
were sacrificed and their serum collected and stored at -80.degree.
C. until analysis. Serum levels of OVA-specific IgE were measured
by sandwich ELISA as described previously (Kanamaru, F., et al.
"Costimulation via Glucocorticoid-Induced TNF Receptor in Both
Conventional and CD25.sup.+ Regulatory CD4.sup.+ T Cells." J
Immunol., Vol. 172, pages 7306-7314 (2004)).
[0161] Bronchoalveolar lavage (BAL) was performed by injection of 1
ml saline (37.degree. C.) through a tracheal cannula into the lung.
Cells in the BAL were centrifuged and resuspended in cold PBS. For
differential BAL cell counts, cytospin preparations were made and
per cytospin, 200 cells were counted and differentiated by standard
morphology and staining characteristics.
[0162] IL-4, IL-5 and IL-13 ELISAs were performed according to the
manufacturer's instructions (R & D systems, Minneapolis,
Minn.). The detection limits of the ELISAs were 60 pg/ml for IL-4,
32 pg/ml for IL-5, 15 pg/ml IL-13.
[0163] The isoproterenol-induced relaxation of tracheal rings from
OVA-sensitized asthmatic animals was also tested. After induction
of asthma, tracheal rings were removed and mounted on the myograph.
Rottlerin (0.5 mM) was added to the bath of each tracheal ring 2
minutes prior to exposure to isoproterenol. At the conclusion of
the experiment, each tracheal ring was exposed to methacholine to
ensure viability of the ring and equivalent constriction. Rottlerin
enhanced the isoproterenol-induced relaxation of the PBS-treated
(control) animals, shifting the isoproterenol-relaxation curve
upward and to the left (FIG. 13). This result indicates that
rottlerin can induce relaxation of tracheal rings in a basal state
(not exposed to methacholine), indicating that there is at least an
additive effect of rottlerin and isoproterenol.
[0164] Trachea obtained from OVA-sensitized animals had a blunted
relaxation response to isoproterenol (FIG. 13). The
isoproterenol-relaxation curve was shifted downward and to the
right, with the maximal relaxation response reduced to only
.about.25% from more than 60%. Remarkably, acute exposure to
rottlerin restored the response to .about.45%, and caused a
leftward shift in the isoproteronol-relaxation curve to
near-normal. These results indicate that rottlerin has a direct and
an acute effect on airway hyper-responsiveness in vitro and these
results laid the foundation for administering rottlerin as a
therapeutic for asthma.
[0165] FIG. 9B shows the increase in airway hyper-responsiveness in
response to increasing doses of MCh. As expected, in response to 25
mg/ml MCh, OVA-treated mice exhibited increased bronchoconstriction
compared to the PBS sensitized mice. Rottlerin-treated mice
sensitized with OVA showed a decrease in airway constriction to
OVA-treated animals at 25 mg/ml methacholine challenge.
[0166] To further confirm these results, airway resistance (RL) in
these animals was measured. In this system mice are anesthetized
and ventilated and through a cannula in the trachea, tracheal
pressure and flow are continuously monitored and traditional
pulmonary mechanics can be measured. This system is preferable over
the method of oscillatory mechanics due to the consistency of
measurements and the ability to directly measure pressure and flow.
Changes in pressure, flow, and volume were recorded, and RL was
calculated from peak values after each challenge (FIG. 14A).
Administration of rottlerin had no significant adverse effect on
any treated mouse--there was no apparent morbidity or any
mortality. Rottlerin-treatment of PBS-sensitized animals had no
effect on airway resistance (FIG. 14A). Indeed, the OVA-sensitized
mice showed an increase in their RL as compared to the
PBS-sensitized mice in response to Mch (25-50 mg/ml). Rottlerin
treated mice exhibited a decrease in their airway resistance (FIG.
14A and FIG. 16B). These results indicate that rottlerin may play a
therapeutic role in attenuating or preventing the development of
asthma.
[0167] To further understand the role of rottlerin, increasing
doses of isoproterenol (i.p. 20, 40 and 100 .mu.g) were
administered to the OVA-sensitized mice (PBS or rottlerin treated)
after the last methacholine-challenge of 50 mg/ml. Rottlerin
treated OVA-sensitized mice showed a significant decrease in airway
constriction as compared to the untreated group, suggesting an
additive effect of rottlerin with isoproterenol (FIG. 14B).
[0168] After determining the airway hyperresponsiveness, lungs were
lavaged with PBS and total cell count with differential was
determined on the bronchoalveolar cells (BAL). Cytospin was
performed on the collected lavage, and cells were stained with Diff
Quik (IMEB, Inc., San Marcos, Calif.). Cell type (e.g.,
eosinophils, marcrophages and lymphocytes) were recognized by
morphometry. The OVA-sensitized mice exhibited an increase in
inflammation and differential count (eosinophils, lymphocytes and
macrophages) (FIG. 16C) (Wang et al., "Endogenous and exogenous
IL-6 inhibit aeroallergen-induced Th2 inflammation" J Immunol
165:4051-4061 (2000)). FIG. 10A shows inflammatory leukocytes
recruited into the lungs following sensitization and challenge with
OVA. OVA-sensitized mice showed an increase in the number and
variety of cells, including macrophages, eosinophils, and
neutrophils as compared to the unsensitized groups. The
OVA-sensitized mice treated with rottlerin showed a significant
decrease in the number of inflammatory leukocytes with a marked
reduction in the number of macrophages and eosinophils. These
results suggest that rottlerin plays an important role in
preventing the inflammatory response in asthma. To confirm
sensitivity with OVA, specific IgE levels were examined 48 hours
after the last airway challenge. Following systemic OVA
sensitization and challenge, there was a significant increase in
the serum IgE levels in the OVA sensitized groups (OVA;
OVA+rottlerin) (FIG. 10B). The serum IgE in the OVA and
OVA+rottlerin groups were similar demonstrating that the animals
were similarly sensitized. The serum IgE levels in both these
groups were approximately equivalent, demonstrating that the
animals were equally sensitized. Consistent with the observed
changes in R.sub.L in response to MCh and the reduction in
inflammatory cells in the BALF in response to rottlerin, a
significant reduction in the Th2 cytokine production was observed
in the BALF of the rottlerin-treated animals. All Th2 cytokines
evaluated, IL-4, IL-5, and IL-13, were significantly increased in
the BALF of the OVA-sensitized animals (FIG. 10C). However, the
production of all these Th2 cytokines was significantly reduced in
the BALF of rottlerin-treated OVA-sensitized animals. There was no
significant difference between the PBS- and rottlerin-treated
control groups.
[0169] Consistent with the observed changes in airway resistance in
response to methacholine and the reduction in inflammatory cells in
the BAL fluid in response to rottlerin, a significant reduction in
the cellular infiltrate in the peribronchial and perivascular
regions was observed in rottlerin-treated OVA-sensitized/challenged
animals compared to PBS-treated OVA-sensitized/challenged animals
(FIG. 17).
[0170] Thus, in the acute asthma model, rottlerin attenuated the
OVA-induced airway hyper-reactivity and pulmonary resistance and
reduced inflammation, demonstrating that rottlerin may play an
important therapeutic role in preventing or attenuating the
development of airway hyperreactivity.
Example 5
Single Dose of Rottlerin Acutely Relaxes OVA-Induced AHR
[0171] Based upon the proposed role for BK channels in modulating
airway contractility, the inventors hypothesized that acute
administration of rottlerin, like .beta.2 adrenergic agonists, can
cause bronchodilatation. An acute asthma model in OVA-sensitized
mice established and validated, as assessed by measuring airway
resistance (R.sub.L) in response to methacholine (MCh). Groups of
mice received an I.P. injection of OVA/Alum complex on days 0 and 7
and on alternate days 14-22, a 20 minute aerosol challenge of
either PBS or 2% (w/v) OVA in PBS, using an ultrasonic nebulizer
(FIG. 22A). The asthma model exhibited an increase in AHR as shown
by an increase in R.sub.L in response to MCh (FIG. 22B). To
determine whether rottlerin could reverse AHR in OVA-asthmatic
mice, a single dose of rottlerin (5 .mu.g/g) was injected
intravenously (I.V.) 5 minutes before measurements of AHR (FIG.
2A). As expected, the OVA-sensitized mice exhibited an increase in
R.sub.L as compared to controls in response to MCh (0-50 mg/ml).
However, the OVA-sensitized group that received rottlerin I.V.
showed a significant decrease in their R.sub.L when compared to the
untreated groups (FIG. 2B). These results indicate that acute
treatment of rottlerin can reverse OVA-induced AHR.
[0172] Control and OVA-challenged mice were treated with rottlerin,
given through the tail vein 5 minutes prior to airway pressure
measurements. Rottlerin significantly reduced airway resistance in
the OVA-sensitized/challenged animals (FIG. 18A), compared to
PBS-treated OVA sensitized/challenged animals. This acute effect is
unlikely to be due to an effect on inflammation given the short
period of time between injection of the drug and measurement of
airway resistance, strongly suggesting a direct effect of rottlerin
on smooth muscle contractility, likely by activating BK channels
and hyperpolarizing membrane potential. Acute administration of
rottlerin, in combination with Isoproterenol, increased
.beta.-agonist mediated relaxation of the airway in OVA-challenged
asthmatic mice (FIG. 18B).
[0173] Next, whether rottlerin can restore isoproterenol
(ISO)-induced relaxation of tracheal rings in OVA-sensitized
asthmatic animals was tested. After induction of asthma, tracheal
rings were removed and mounted on the myograph. Rottlerin (0.5
.mu.M) was added to the bath of each tracheal ring 2 minutes prior
to exposure to ISO. As seen in FIG. 21B, tracheas obtained from
OVA-sensitized animals had a blunted relaxation response to ISO.
The ISO-relaxation curve was shifted downward and to the right,
with the maximal relaxation response reduced to only .about.25%
from more than 60%. Remarkably, acute exposure to rottlerin in the
bath restored the response to .about.45%, and caused a leftward
shift in the ISO-relaxation curve to near-normal. These results
indicate that rottlerin has a direct and an acute effect on airway
hyper-responsiveness in vitro.
Example 6
House Dust Mite Antigen Induction of Asthma
[0174] Whether rottlerin could affect AHR in the house dust mice
antigen model of asthma was determined. House dust mite (HDM) is
one of the most common aeroallergens and is implicated in allergy
and asthma symptoms in .about.10% of the population (Johnson et
al., "Continuous exposure to house dust mite elicits chronic airway
inflammation and structural remodeling." Am J Respir Crit Care Med
169:378-385 (2004)). Exposure to HDM extract elicits a severe and
persistent eosinophilic airway inflammation. Mice were exposed to
purified HDM extract (Greer Laboratories, Lenoir, N.C.)
intranasally (25 .mu.g of protein in 10 .mu.l saline) for 5
days/week for 3 weeks as previously described (Id.). Rottlerin (5
.mu.g/g=100 .mu.g/mouse intraperitoneal) was administered every
other day (see protocol in FIG. 19A). Changes in pressure, flow,
and volume were recorded, and airway resistance was calculated from
peak values after each challenge using a Buxco forced maneuvers
system and restrained whole body plethysmography (rWBP; PLY3011
chamber, Buxco (BUXCO Electronics Inc., Troy, N.Y.)).
Administration of rottlerin had no significant adverse effect on
any treated mouse--there was no increase in mortality or apparent
morbidity in rottlerin-treated animals. Rottlerin-treatment of
PBS-sensitized animals had no effect on airway resistance
[0175] As expected, the HDM-exposed mice exhibited an increase in
airway resistance as compared to PBS-sensitized mice in response to
methacholine (25-50 mg/ml) (FIG. 19B). The rottlerin-treated,
HDM-sensitized mice exhibited a marked decrease in their airway
resistance when compared to HDM-sensitized, PBS-treated animals
(FIG. 19B). Consistent with the observed changes in airway
resistance in response to methacholine, a significant reduction in
the cellular infiltrate was observed in the peribronchial and
perivascular regions in rottlerin-treated HDM-exposed animals
compared to PBS-treated HDM-exposed animals (FIG. 20).
[0176] The predominant features of asthma are: A) the inappropriate
constriction of airway smooth muscle (ASM), and B) inflammation.
The contractility of airway smooth muscle is regulated in part by
plasma membrane BK channels (large conductance voltage- and
Ca.sup.2+-activated K.sup.+ channels). BK channel activation causes
transient membrane hyperpolarization, inhibition of Ca.sup.2+
influx through voltage-dependent Ca.sup.2+ channels, reduced
[Ca.sup.2+].sub.i and smooth muscle relaxation. Evidence supporting
a role for BK channels in modulating airway contractility is based
upon animal and human studies in which reduction in BK channel
function is associated with airway hypercontractility. Moreover,
polymorphisms in the BK channel have been identified that are
associated with more severe forms of asthma in humans. The
inventors have shown that a small molecule, rottlerin, directly and
potently activates BK channels. Systemic administration of
rottlerin, both acutely and chronically, attenuates the induction
of airway hyperreactivity in two models of asthma: (1) ovalbumin
exposure model; (2) house dust mice exposure model. In addition,
the inventors showed that rottlerin potently inhibits the
immunological response, with a marked reduction in peribronchial
and perivascular infiltration of immune cells. Moreover, the
inventors demonstrated that a single injection of rottlerin via the
tail vein of mice can significantly diminish methacholine-induced
airway hyperreactivity in the ovalbumin asthma model. The effects
on reducing airway hyperreactivity are likely mediated both through
BK channel-dependent effects on smooth muscle relaxation and
through BK channel-independent effects on immunologic mediators of
asthma. By targeting BK channels, which are not expressed in the
heart, airway smooth muscle reactivity may be normalized without
the side-effects observed with standard therapies. This approach is
entirely novel as it combines anti-inflammatory actions with direct
airway smooth muscle relaxation in a single therapeutic.
Example 7
Dosage Determination and Aerosolized Delivery of Rottlerin
[0177] The data above demonstrate that systemic (I.P.)
administration of rottlerin for 2 weeks during OVA or HDM challenge
markedly attenuates AHR, and inflammation in OVA and HDM sensitized
mice. Whether aerosolized delivery of the drug is effective in
these two asthma models will be tested. Aerosolized delivery has
the advantages of: 1) direct delivery to the target organ which has
a large absorptive surface and eliminates first pass metabolic
degradation; 2) potentially reducing systemic adverse effects (e.g.
lowering BP); 3) rapid onset of action. For these reasons most
currently effective therapies for asthma including steroids, and
bronchodilators are delivered using aerosolized forms (Sears et
al., "Regular inhaled beta-agonist treatment in bronchial asthma."
The Lancet, 1990. 336(8728): p. 1391-1396; Barnes, P. J., "Inhaled
glucocorticoids for asthma." N Engl J Med, 1995. vol. 332(13): p.
868-75.)
[0178] Ultrasonic nebulization for the airway delivery of rottlerin
will be used as described (Hrvacic et al., "Applicability of an
ultrasonic nebulization system for the airways delivery of
beclomethasone dipropionate in a murine model of asthma." Pharm
Res, 2006. 23(8): p. 1765-75; Wiedmann, T. S. and A. Ravichandran,
Ultrasonic nebulization system for respiratory drug delivery. Pharm
Dev Technol, 2001. 6(1): p. 83-9). Ultrasonic nebulizers produce
aerosols from drug solutions by converting electrical pulses to
mechanical vibrations (Atkins, P. and A. R. Clark, Drug delivery to
the respiratory tract and drug dosimetry. J Aerosol Med, 1994.
7(1): p. 33-8). These nebulizers have been previously used for the
inhalation delivery of anti-asthma drugs in murine models of asthma
(Hrvacic et al., "Applicability of an ultrasonic nebulization
system for the airways delivery of beclomethasone dipropionate in a
murine model of asthma." Pharm Res, 2006. 23(8): p. 1765-75).
Microsuspensions of rottlerin will be aerosolized using a nebulizer
and delivered to mice via a nose-only inhalation route; the total
dose will be controlled by varying formulation strength while
holding exposure time constant.
[0179] To determine basic pharmacokinetic profiles, mice will be
exposed to aerosols for a single 30-minute period; then, 30 minutes
post-treatment, lung tissues, plasma, and BALF will be analyzed for
rottlerin content. The aerosolized rottlerin will be administered
for 5 or 21 days and lung tissues, plasma, and BALF will be
analyzed for rottlerin content to determine whether steady state
levels are achieved and what the tissue half-life [t1/2] of
rottlerin is. To correlate these exposure levels with safety,
animals will be monitored for changes in blood pressure, weight,
and heart rate. Relevant organs including lungs, heart, liver,
kidneys, and digestive tract will be examined for histological
changes after treatment compared with organs taken from untreated
mice. Aerosolized rottlerin (molecular formula:
C.sub.30H.sub.28O.sub.8, MW: 516) will be prepared according to
published protocols used for compounds of similar molecular weight
and solubility. Rottlerin is soluble to 2 mM in ethanol and to 100
mM in DMSO. The ultrasonic nebulization system (UNS) that will be
used consists of an ultrasonic nebulizer, drying column, deionizer
and animal exposure chamber. Solutions will be pumped into the
nebulizer with a syringe pump. The nebulizer and an ultrasonic
spray nozzle system will be driven by an ultrasonic generator
operating at a fixed frequency of 125 kHz attached to an air supply
device that injects a stream of air (3 l/min) around nozzle of the
nebulizer mounted on the upper conical portion of a 500 ml
round-bottom flask connected to a 45 cm long drying column using
tygon tubing. Spray drying of the aerosol is accomplished by
passage through the inner cylinder of drying column surrounded by
charcoal. The outlet of the drying column is connected through the
deionizer to an animal exposure chamber. Using the established
asthma protocols, OVA, HDM and control (PBS) mice will receive a 20
minute aerosol treatment with either DMSO (0.1% in PBS) or
rottlerin (0.1, 1, 10 and 100 .mu.g/g in a total volume of 150
.mu.l PBS) the dose administered to the mice will be estimated
according to the formula described (Wattenberg et al.,
"Chemoprevention of pulmonary carcinogenesis by brief exposures to
aerosolized budesonide or beclomethasone dipropionate and by the
combination of aerosolized budesonide and dietary myo-inositol"
Carcinogenesis, vol. 21(2): p. 179-82 (2000)), using a lumiscope
6610 ultrasonic nebulizer (Lumiscope Co. Inc., Piscataway, N.J.).
Mice will be intubated and R.sub.L measured before and after MCh
challenge. The inflammatory components of asthma (inflammatory
cells and perivascular/peribronchial cellular infiltration) will be
assessed for the untreated and rottlerin-treated asthmatic animals
as described in the preliminary data section. The following will be
determined: 1) the dose-response curve for rottlerin and calculate
the half maximal effective concentration (EC.sub.50); 2) the
effects on blood pressure since BK channels are present in vascular
smooth muscle (Brenner, R., G. J. Perez, A. D. Bonev, D. M. Eckman,
J. C. Kosek, S. W. Wiler, A. J. Patterson, M. T. Nelson, and R. W.
Aldrich, "Vasoregulation by the beta1 subunit of the
calcium-activated potassium channel" Nature, vol. 407(6806): p.
870-6 (2000); Ledoux, J., M. E. Werner, J. E. Brayden, and M. T.
Nelson, "Calcium-activated potassium channels and the regulation of
vascular tone" Physiology (Bethesda), Vol. 21: p. 69-78 (2006)); 3)
whether mucin production is reduced by rottlerin (via mucin
staining of histological sections) as has been reported (Park et
al., "Protein kinase C delta regulates airway mucin secretion via
phosphorylation of MARCKS protein" Am J Pathol. Vol. 171(6): p.
1822-30 (2007)); and 4) the minimal effective dose in OVA and HDM
asthma models. The R.sub.L measured in response to MCh (12.5 mg/ml)
will be plotted against the rottlerin dose to obtain the
dose-response curve which will yield: a) potency, b) maximal
efficacy or ceiling effect (greatest attainable response), c) slope
(change in response per unit dose), d) EC.sub.50 (half maximal
effective concentration), e) minimal effective dose, f) maximal
tolerated dose. These studies will provide the basis for estimating
pharmacokinetics of rottlerin and the design of clinical
trials.
[0180] As an alternative to aerosolized rottlerin, the use of
liposome mediated delivery will be explored. Liposome mediated
delivery has been used successfully to deliver drugs with similar
characteristics to rottlerin to the lungs (Chougule, M., B. Padhi,
and A. Misra, "Nano-liposomal dry powder inhaler of tacrolimus:
preparation, characterization, and pulmonary pharmacokinetics" Int
J Nanomedicine, Vol. 2(4): p. 675-88 (2007); Waldrep, J. C., "New
aerosol drug delivery systems for the treatment of immune-mediated
pulmonary diseases" Drugs Today (Barc), Vol. 34(6): p. 549-61
(1998)).
[0181] To determine whether rottlerin administration after asthma
is established is effective and determine the duration of action of
a single rottlerin administration in two murine asthma models, a
dosing schedule will be used. In this dosing schedule, rottlerin
will be administered every other day, starting on day 12 of a
24-day asthma-induction protocol, just prior to the OVA or HDM
nebulization. A final dose of rottlerin will be given 1 hour prior
to the assessment of AHR on the final day of the asthma induction
protocol. Moreover, the examples above have shown that a single
I.V. administration of rottlerin can acutely (within 5 minutes)
reduce AHR in these models. The goal is to determine whether
rottlerin administered every other day for 1-3 weeks after asthma
has been established attenuates both the inflammatory response and
AHR in the OVA-induced and HDM models of asthma. The effects of
rottlerin on airway remodeling in the HDM model will also be
examined. In addition, the duration of action of a single
administration of aerosolized rottlerin will be determined.
[0182] The details of the dosage schedule for rottlerin is as
follows. Once the optimal dose of aerosolized rottlerin in OVA and
HDM murine asthma models and controls (PBS sensitized) is
identified, this dose will be administered after the induction of
asthma (vs. administration of rottlerin during the asthma induction
protocol). In these experiments, rottlerin treatment will be
initiated after 3 weeks of OVA sensitization and after 5 weeks of
HDM sensitization. OVA and HDM challenges will be continued and
animals will be treated for 1-3 weeks with the optimal dose of
aerosolized rottlerin. AHR will be determined prior to sacrifice.
Histological analyses of inflammation and airway remodeling, and
BAL cell counts will be performed. Serum and tissue samples will be
obtained to determine the rottlerin drug levels in these
aerosolized treated animals using previously reported techniques
(Varma et al., "Oral contraptive--Part III. Further observations on
the antifertility effect of rottlerin" Indian J Physiol Pharmacol.
Vol. 3: p. 168-72 (1959)). If for any reason aerosolized rottlerin
is not efficacious, the dosing experiments will be performed using
systemic administration.
[0183] The duration of action of a single administration of
aerosolized rottlerin will be determined by measuring the R.sub.L
in response to MCh at 5, 30, 60, 120 minutes and 6 and 12 hours
following the drug administration to determine duration of action
of rottlerin in the OVA and HDM mouse asthma models.
Example 8
Rottlerin Derivatives
[0184] One approach to understand the mechanism by which rottlerin
attenuates AHR and inflammation is to dissociate the two effects by
developing rottlerin derivatives, some of which may lack BK
modulatory properties or lack anti-inflammatory properties. Two
derivatives, reduced rottlerin and methylated rottlerin, are shown
in FIG. 24. Methylated rottlerin is an inhibitor of BK channel,
whereas reduced rottlerin is an activator, albeit less potent than
rottlerin, of BK channels. Methylated rottlerin significantly slows
activation kinetics (time for opening) of the channel and shifted
the G-V curve to the right compared to control (FIG. 24E). In
contrast, reduced rottlerin shifts the G-V curve to the left
compared to control (FIG. 24F), although the shift is significantly
less than rottlerin (FIG. 24D).
[0185] Rottlerin derivatives will be compared with rottlerin. The
ones with higher specificity for BK channels (i.e. BK activating
properties without effects on other ion channels or on T cells) and
ones with higher specificity for T-cell suppression (i.e. no effect
on BK channels or other ion channels) will be identified. In
addition to specificity, derivatives with higher potency for either
or both of the two activities will also be identified. The most
promising derivatives in terms of specificity and/or potency will
be advanced to animal studies to evaluate the separate therapeutic
contributions of BK activation and T-cell suppression in the
application of rottlerin and its derivatives to the alleviation of
asthma. In this way, the basis for the alleviation of asthma by
rottlerin and its derivatives will be better understood and
important therapeutic lead compounds will be found.
[0186] In testing rottlerin derivatives for inducing membrane
hyperpolarization via activation of BK channels, the Molecular
Devices FLIPR system and the membrane potential assay kit (cat
#R8034) (Molecular Devices, Sunnyvale, Calif.) will be used. The
system has been used to perform high throughput screening of
K.sup.+ channel activators (Vasilyev et al., A novel
high-throughput screening assay for HCN channel blocker using
membrane potential-sensitive dye and FLIPR. J Biomol Screen, 14(9):
p. 1119-28 (2009)). The assay is based on the use of fluorescent
dyes, which accumulate inside cells upon depolarization of the
membrane potential, leading to elevated fluorescence.
Hyperpolarization of the membrane potential leads to reduced
fluorescence. HEK cells stably expressing human BK channels will be
plated at 2.6.times.10.sup.4 cells/well (96-well plate)--this
plating density results in full confluency of cells in the plate 24
hours post-plating. Cell viability will be confirmed by propidium
iodide exclusion. Growth media will be removed from the microplate
using a Microplate Washer (BioTek Instruments Inc., Winooski, Vt.)
and the cells washed with Hank's balanced salt saline (HBSS). The
cells will be loaded with the membrane potential sensitive dye at
room temperature for 30 minutes. The solution will be aspirated
leaving only residual volume and the plates positioned within the
FLIPR reading chamber. Background fluorescence will be monitored
for 14 seconds followed by a single step addition of rottlerin or
its derivatives, in varying concentrations. The fluorescence
response will be captured for 5 minutes in 2-second intervals. At
the conclusion of the 5-minute period, iberiotoxin, a specific BK
channel inhibitor, will be added. For all experiments, incubation
with iberiotoxin will also be included during the dye loading step
as a control. In these controls, no change in fluorescence should
be observed upon addition of rottlerin or its derivatives. Data
will be analyzed using FLIPR software, in which % reduction in
fluorescence (which is associated with membrane hyperpolarization)
will be plotted against the respective drug concentrations
(Vasilyev et al., A novel high-throughput screening assay for HCN
channel blocker using membrane potential-sensitive dye and FLIPR. J
Biomol Screen, 14(9): p. 1119-28 (2009)) and compared to
rottlerin.
[0187] Whether rottlerin derivatives activate hERG, leading to
membrane hyperpolarization will be determined. A similar approach
using voltage-sensitive dyes has been previously used to determine
hERG channel inhibition (Dorn et al., Evaluation of a
high-throughput fluorescence assay method for HERG potassium
channel inhibition. J Biomol Screen, 10(4): p. 339-47 (2005)). A
CHO-hERG stable cell line will be used. The voltage sensitive
oxonol dyes, such as DiBAC.sub.4, at concentrations of 10 nM and
higher significantly increase activity of BK channels in the
presence of the .beta.1 subunits (Morimoto et al.,
Voltage-sensitive oxonol dyes are novel large-conductance
Ca.sup.2+-activated K.sup.+ channel activators selective for
.beta.1 and .beta.4 but not for .beta.2 subunits. Mol Pharmacol,
71(4): p. 1075-88 (2007)). BK .alpha. subunit alone, however, is
not affected by up to 1 .mu.M DiBAC.sub.4. Thus, since rottlerin's
activation of BK channels is not dependent upon .beta. subunits,
the derivatives will be tested with the FLIPR system on channels
composed of BK .alpha. subunit only.
[0188] Rottlerin derivatives will be tested for BK channel function
(electrophysiology and tracheal rings). For those compounds with
comparable EC.sub.50 as rottlerin, cellular electrophysiology
studies will be perform using patch clamp of BK .alpha.
subunit-expressing-HEK cells (stable line) and acutely isolated
tracheal smooth muscle. The effects of the rottlerin derivatives on
the rates of opening and closing of the channel and
conductance-voltage relationship will be studied (Zakharov et al.,
Activation of the BK (SLO1) potassium channel by mallotoxin. J Biol
Chem, 280(35): p. 30882-7 (2005)). For electrophysiological testing
in isolated ASM, physiological changes in BK channel activity are
assessed by measuring spontaneous transient outward currents
(STOCs) in non-dialysed cells by perforated patch-clamp recordings.
STOCs are BK channel openings caused by instantaneous ryanodine
receptor openings. STOC amplitude represents the number of BK
channels opening after a spark event (Zhuge et al., Spontaneous
transient outward currents arise from microdomains where BK
channels are exposed to a mean Ca.sup.2+ concentration on the order
of 10 microM during a Ca.sup.2+ spark. J Gen Physiol, 2002. 120(1):
p. 15-27). STOCs (perforated patch, voltage steps from -70 mV,
stepped at 10 second intervals to +30 mV) will be measured in
isolated tracheal ASMC.
[0189] Rottlerin derivatives will also be tested for relaxation of
murine tracheal rings. A similar approach as the one in Example 4
(see also FIG. 13), in which rottlerin enhanced
isoproterenol-mediated relaxation in trachea derived from both
control and OVA-sensitized/challenged animals, will be taken.
[0190] An important finding is the marked diminution of
inflammatory cells in the BAL fluid and in the peribronchial and
perivascular space in the rottlerin-treated asthmatic mice as
compared to PBS-treated asthmatic mice. The inventors have found
that rottlerin significantly decreases the Th2 cytokines, IL-4 IL-5
and IL-13 in the BAL fluid of asthmatic mice. Rottlerin is known to
inhibit human T cell responses (Springael et al., Rottlerin
inhibits human T cell responses. Biochem Pharmacol, 73(4): p.
515-25 (2007)), and PMA-induced phosphorylation of Erk-1 and Erk-2
in Jurkat T cells and purified human CD4+ T cells from peripheral
blood (Roose et al., A diacylglycerol-protein kinase C-RasGRP1
pathway directs Ras activation upon antigen receptor stimulation of
T cells. Mol Cell Biol, 25(11): p. 4426-41 (2005)).
[0191] Thus, rottlerin and its derivatives will be tested for
immunological effect using an in vitro lymphocyte assay. The
OVA-specific responses in thoracic lymph node cultures will be
examined as previously described (Bao et al., A novel
antiinflammatory role for andrographolide in asthma via inhibition
of the nuclear factor-kB pathway. Am J Respir Crit Care Med, 179:
p. 657-665 (2009)). The thoracic lymph nodes will be harvested from
mice 24 hours (Lai et al, The role of sphingosine kinase in a
murine model of allergic asthma. J Immunol, 180: p. 4323-4329
(2008)) after the last OVA aerosol challenge. Lymph node cultures
will be exposed to 200 .mu.g/ml OVA for 72 hours in the absence or
presence of rottlerin (doses 0.1, 1, 10, 50 .mu.M) or derivatives.
The levels of IL-4, IL-5 and IFN-.gamma. in culture supernatant
will be determined using ELISA.
[0192] Certain derivatives will be further studied in vivo to
assess the relative contributions of BK channel activation and
anti-inflammatory effects to the rottlerin-induced reduction in AHR
observed in the OVA-asthma model. The derivatives to be studied in
vivo will be categorized based upon their efficacy as a BK channel
agonist (without anti-inflammatory effects), BK channel agonist
with anti-inflammatory effects and an anti-inflammatory effects
without BK channel activating properties. Selected derivatives
administered, systemically or via nebulization, acutely or over an
extended period, will be studied.
[0193] The derivatives will be compared to rottlerin and PBS in the
mice subjected to the 24 day OVA-asthma model as described above,
to determine whether specific BK channel agonists can acutely
dilate hypercontractile airway and to dissect the molecular
mechanisms underlying the rottlerin-induced attenuation in airway
hyperreactivity in the murine asthma model. The derivatives will be
injected I.P. every other day and airway resistance will be
determined, in response to methacholine at day 24. Cell counts and
analysis of cytokines (levels of Th-2 type cytokines and Th-1 type
cytokine, IFN-.gamma.,) will be determined in peripheral blood and
BAL fluid.
[0194] The following methodologies may be used to perform the
experiments outlined above.
Smooth Muscle Cell Isolation
[0195] Mice will be euthanized by injection of sodium
pentobarbital, trachea removed and transferred to ice-cold-low
Ca.sup.2+ physiological saline solution (PSS). After the removal of
epithelium, cartilage and connective tissue, the trachealis muscle
will be minced and placed in PSS containing papain, DTT and bovine
serum albumin at 37.degree. C. for 20 minutes, followed by PSS
containing collagenase H, collagenase II, DTT and BSA at 37.degree.
C. for 30 minutes. The digested tissue will be washed, and single
cells released by gentle trituration with a fire-polished glass
pipette.
Cellular Electrophysiology
[0196] Spontaneous BK currents will be measured using the
whole-cell patch clamp technique in the amphotericin B (250
.mu.g/ml) perforated patch configuration as described by Santana
and colleagues (Amberg et al., Modulation of the molecular
composition of large conductance, Ca.sup.2+ activated K.sup.+
channels in vascular smooth muscle during hypertension. J Clin
Invest, 112(5): p. 717-24 (2003); Amberg, G. C. and L. F. Santana,
Downregulation of the BK channel beta1 subunit in genetic
hypertension. Circ Res, 93(10): p. 965-71 (2003)). Cells will be
continuously superfused with normal Tyrode's solution. ASMC will be
held at -40 mV. Petri dishes with ASMC cells will be mounted on the
stage of an inverted microscope, which will serve as a perfusion
chamber. Experimental solutions will be applied by local
perfusion.
In Vivo Measurement of Airway Hyperreactivity (AHR)
[0197] Two days after the last aerosol, AHR will be measured by
invasive restrained whole body plethysmography (rWBP) (BUXCO
Electronics) in response to inhaled methacholine (Sigma, St. Louis,
Mo.). For dynamic lung resistance measurements will be performed
using the PLY3011 chamber (BUXCO). Mice will be anesthetized with a
cocktail of 25 mg per kg body weight ketamine hydrochloride
(Bioniche Pharma USA LLC, Lake Forest, Ill.) and 2.5 mg per kg body
weight xylazine (Lloyd Labs, Quezon City, Philippines),
tracheotomized, and immediately intubated with an 18-gauge
catheter, followed by mechanical ventilation (Columbus Instruments,
Columbia, Ohio). Respiratory frequency will be set at 150
breaths/minute with a tidal volume of 0.2 ml. Increasing
concentrations of methacholine (0-50 mg/ml) will be administered at
the rate of 20 puffs per 10 seconds, with each puff of aerosol
delivery lasting 10 ms, via a nebulizer aerosol system with a 4-6
.mu.m aerosol particle size generated by a nebulizer head (Aeroneb,
Aerogen Ltd., Galway, Ireland). The nebulizer will be attached to a
small aerosol block, which is placed in the inspiratory flow line.
In this way the aerosol will be injected directly into the airway.
Baseline resistance will be restored before administration of the
subsequent doses of methacholine. The flow and pressure signals
will be measured and processed together to determine resistance and
compliance using BioSystem XA software (BUXCO).
Other OVA Models
[0198] The intermediate and long-term OVA models may also be used
to explore the efficacy of rottlerin and rottlerin derivative.
[0199] All documents cited above are incorporated by reference as
if recited in full herein.
[0200] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
* * * * *